The Naturally Occurring A51V Variant of Human Cytochrome c Destabilizes the Native State and Enhances Peroxidase Activity

Abstract

The A51V variant of human cytochrome c is linked to thrombocytopenia 4 (THC4), a condition that causes decreased blood platelet counts. A 1.82 Å structure of the A51V variant shows only minor changes in tertiary structure relative to the wild type (WT) protein. Guanidine hydrochloride denaturation demonstrates that the global stability of the A51V variant is 1.3 kcal/mol less than that of the WT protein. The midpoint pH, pH_1/2, of the alkaline transition of the A51V variant is 1 unit less than that of the WT protein. Stopped-flow pH jump experiments show that the A51V substitution affects the triggering ionization for one of two kinetically distinguishable alkaline conformers and enhances the accessibility of a high spin heme transient. The pH_1/2 for acid unfolding of the A51V variant is 0.7 units higher than for that of the WT protein. Consistent with the greater accessibility of non-native conformers for the A51V variant, the k_cat values for its peroxidase activity increase by 6 to 15 fold in the pH range 5 to 8 versus those of the WT protein. These data along with previously reported data for the other THC4–linked variants, G41S and Y48H, underscore the role of Ω-loop C (residues 40 – 57) in modulating the peroxidase activity of cytochrome c early in apoptosis.

Graphical Abstract

INTRODUCTION

The role of mitochondrial cytochrome c (Cytc) as an electron carrier in the electron transport chain is well-established.¹ More recently Cytc has been shown to have diverse cellular functions,² and in particular plays an important role in the intrinsic apoptotic pathway.^3–4 Under apoptotic stimuli, Cytc can oxygenate polyunsaturated acyl chains of the mitochondrial lipid cardiolipin (CL)^5–6 leading to Cytc dissociation from the membrane. Upon passage from the mitochondria into the cytoplasm, Cytc interacts with apoptotic protease activating factor 1, Apaf-1, to form a complex known as the apoptosome. The apoptosome cleaves procaspase-9 to its active form, caspase-9, ultimately leading to cell death.⁷

Cytc contains a single covalently attached heme group. Cys14 and Cys17 bind the heme via two thioether bonds, while His18 and Met80 are the axial ligands of the heme iron.⁸ Thus, the heme iron is six-coordinate and low spin. Heme ligation remains the same in both ferricytochrome c and ferrocytochrome c, making Cytc an efficient electron transporter. When Cytc interacts with CL, Cytc can become a peroxidase leading to oxygenation of CL.⁵ It is known that heme peroxidases need a pentacoordinate heme or an available heme coordination site to allow the Fe(III) to react with H₂O₂.⁹ Thus, to convert Cytc from an electron carrier to a classical heme peroxidase, the heme ligation of Cytc needs to be changed. Previously, we have demonstrated that modest structural rearrangement of Ω-loop D permits loss of Met80-heme ligation allowing Cytc to gain peroxidase function.¹⁰ We have also shown that the hydrocarbon chains of detergents can act as structural mimics for CL. These detergents facilitate dimerization of iso-1-Cytc to create an available heme coordination site,¹¹ leading to enhancement of the peroxidase activity of Cytc.¹²

Given the essential biochemical functions of Cytc, few naturally occurring variants have been documented in humans. Thrombocytopenia 4 (THC4; OMIM 612004) is an inherited autosomal-dominant disease whose symptom is a decrease in the number of platelets in circulating blood.^13–14 THC4 is caused by mutation in the Cytc gene (CYCS). Three Cytc variants (G41S, Y48H and A51V) have been identified so far that cause THC4.^13–16 G41S and Y48H human (Hu) Cytc have been extensively studied.^17–24 Both the G41S and Y48H variants of Hu Cytc show increased flexibility of Ω-loop C (residues 40 – 57) and Ω-loop D (residues 71 – 85)^19–20 as well as more accessible alkaline conformers, consistent with the enhanced peroxidase activity of both variants. Interestingly, all three naturally occurring mutations are located in Ω-loop C, which is the least stable substructure of Cytc.²⁵ Recent in vivo studies indicate that the enhanced peroxidase activity of the THC4-linked Cytc variants is unlikely the cause of THC4.¹⁴ However, these variants remain excellent model systems for probing the structural factors that control the peroxidase activity that mediates the early stages of apoptosis.⁵

In the present study, we probe the effect of the naturally occurring A51V variant on the properties of Hu Cytc. We present an X-ray structure of A51V Hu Cytc and characterize the global stability, the local stability and the dynamics of Ω-loop D as measured by the alkaline conformational transition, the acid unfolding and the peroxidase activity of A51V Hu Cytc.

EXPERIMENTAL METHODS

Mutagenesis and Protein Purification.

A51V and A51V-r mutagenesis primers (Invitrogen; see Table S1) were used to add the A51V mutation via PCR-based mutagenesis (QuikChange Lightening, Agilent) to the WT Hu Cytc gene in the pBTR(HumanCc) plasmid.²⁶ The pBTR(HumanCc) vector is a derivative of the pBTR1 expression vector^27–28 with the yeast iso-1-Cytc gene replaced with a synthetic Hu Cytc gene. It co-expresses yeast heme lyase allowing covalent attachment of heme to the CSQCH heme recognition sequence of Hu Cytc in the cytoplasm of Escherichia coli. Sequencing to confirm the mutation required for the A51V variant was performed by Eurofins Genomics (Louisville, KY).

The A51V variant of Hu Cytc was expressed in BL21(DE3) E. coli cells (BioRad, phage T1 resistant strain) transformed with the A51V pBTR(HumanCc) plasmid. Purification was carried out as previously reported.^29–32 Briefly, cells were broken using a Q700 sonicator (Qsonica, LLC), and the lysate was cleared via centrifugation. Following 50% ammonium sulfate saturation, precipitates were removed via centrifugation, and the supernatant was dialyzed against 12.5 mM sodium phosphate, pH 7.2, 1 mM EDTA, 2 mM β-mercaptoethanol (β-ME). Protein was then batch absorbed to CM-Sepharose Fast Flow resin pre-equilibrated to 50 mM sodium phosphate buffer, pH 7.2, 1 mM EDTA, 2 mM β-ME, and then eluted with a linear gradient of 0 – 0.8 M NaCl in 50 mM sodium phosphate buffer, pH 7.2, 1 mM EDTA, 2 mM β-ME. After concentration and exchange into 50 mM sodium phosphate, pH 7, by ultrafiltration, 1.5 mL aliquots of ~3 mg/mL protein were flash frozen in liquid nitrogen and stored at −80 °C. Prior to experiments, proteins were purified using a HiTrap SP HP 5.0 mL column coupled to an ÄKTAprime plus chromatography system (GE Healthcare Life Sciences). Protein samples were concentrated by ultrafiltration and oxidized with K₃[Fe(CN)₆]. Separation of oxidized Cytc from the oxidizing agent was accomplished by Sephadex G25 chromatography using a running buffer appropriate to the planned experiment.

Global Stability Measurements by Guanidine Hydrochloride Denaturation.

Global stability measurements were performed using guanidine hydrochloride (GdnHCl) as a denaturant. Measurements were performed with an Applied Photophysics Chirascan circular dichroism (CD) spectrometer interfaced with a Hamilton Microlab 500 Titrator at 25 °C, as previously discussed.^29–33 Briefly, the A51V variant at 4 μM in 20 mM Tris, pH 7.5, 40 mM NaCl and ~6 M GdnHCl was titrated into protein at the same concentration in 20 mM Tris, pH 7.5, 40 mM NaCl in a 4 mm pathlength cuvette (Hellma Analytics, Part No. 119004F-10–40) containing a stir bar. After each addition, the sample was stirred to mix, followed by data collection at 222 and 250 nm. Baseline correction was accomplished by subtracting the ellipticity at 250 nm from the ellipticity at 222 nm (θ_222corr = θ₂₂₂ − θ₂₅₀). Plots of θ_222corr versus GdnHCl concentration for A51V Hu Cytc were fit to a two-state model using nonlinear least-squares methods (SigmaPlot v. 13; Systat Software, Inc.), assuming a linear free energy relationship and with native and denatured state baselines that depend linearly on GdnHCl concentration, eq 1.³⁴

$${\text{θ}}_{222\text{corr}}=\frac{\left[\left({\theta }_{\text{N}}+m_{\text{N}}\left[\text{GdnHCl}\right]\right)+\left({\theta }_{\text{D}}+m_{\text{D}}\left[\text{GdnHCl}\right]\right)\cdot \text{exp}\left\{\frac{m\left[\text{GdnHCl}\right]-\mathrm{\Delta }{\text{G}}_{\text{u}}^{°\text{'}}\left({\text{H}}_2\text{O}\right)}{\text{RT}}\right\}\right]}{1+\text{exp}\left[\frac{m\left[\text{GdnHCl}\right]-\cdot {\text{G}}_{\text{u}}^{°\text{'}}\left({\text{H}}_2\text{O}\right)}{\text{RT}}\right]}$$ (1)

In eq 1, θ_N and m_N are the intercept and the slope of the native state baseline, θ_D and m_D are the intercept and the slope of the denatured state baseline, m is the rate of change (slope) of ΔG_u with respect to GdnHCl concentration and ΔG_u^o’(H₂O) is the free energy of unfolding extrapolated to 0 M GdnHCl. ΔG_u^o’(H₂O) and m are used to calculate the concentration of GdnHCl at the unfolding midpoint, C_m (= ΔG_u^o’(H₂O)/m). Reported parameters are the average and standard deviation of three independent trials.

Measurement of the Alkaline Conformational Transition.

A Beckman Coulter DU 800 spectrophotometer was used for pH titrations monitored at 695 nm and 22 ± 2 °C to measure the alkaline conformational transition, as previously described.³⁵ Briefly, a 600 μL protein stock solution of ~200 μM oxidized A51V Hu Cytc in 200 mM NaCl was prepared. The protein stock solution and Milli-Q water were mixed 1:1 to produce a working solution of ~100 μM oxidized A51V in 100 mM NaCl. pH titrations were carried out by adding equal volumes of either NaOH or HCl solutions of appropriate concentration and the protein stock solution so that protein concentration in the working solution did not change during the titration. pH was measured with a Denver Instrument UB-10 pH/mV meter using an Accumet double junction semi-micro pH probe (Fisher Scientific, Cat. No. 13-620-852). Absorbance at 750 nm was subtracted from absorbance at 695 nm to correct for baseline drift (A_695corr = A₆₉₅ − A₇₅₀) during the titration. A_695corr was converted to an extinction coefficient, ε_695corr, by dividing by protein concentration determined using the published extinction coefficients for the isosbestic points at 526.5 and 541.75 nm for spectra near neutral pH.³⁶

Plots of ε_695corr versus pH for the A51V variant were fit to a modified form of the Henderson−Hasselbalch equation, eq 2.

$${\text{ε}}_{695\text{corr}}=\frac{{\text{ε}}_{\text{N}}+{\text{ε}}_{\text{Alk}}\times {10}^{n\left[{\text{pH}}_{1/2}-\text{ pH}\right]}}{1+{10}^{n\left[{\text{pH}}_{1/2}-\text{ pH}\right]}}$$ (2)

In eq 2, ε_N is the corrected extinction coefficient at 695 nm for the native state with Met80 bound to the heme, ε_alk is the corrected extinction coefficient at 695 nm for the alkaline state with lysine as the alkaline state heme ligand,^{28, 37} pH_1/2 is the midpoint pH of the alkaline transition, and n is the number of protons linked to the alkaline transition.

Acid unfolding of A51V Hu Cytc.

Acid unfolding of the A51V variant of Hu Cytc was monitored in the 500 – 750 nm spectral region with a Beckman Coulter DU 800 spectrophotometer. The initial sample was prepared as for the alkaline conformational transition. A protein stock solution of ~200 μM oxidized protein in 200 mM NaCl was prepared and mixed with an equal volume of MilliQ water producing a ~100 μM working solution of the A51V variant in 100 mM NaCl. The working solution was titrated from pH 7.0 to pH 2.0 by adding equal volumes of HCl solutions of appropriate concentration and the protein stock solution so that the protein concentration remained unchanged in the working solution during the titration. After each addition, a spectrum was acquired and pH was measured with a Denver Instrument UB-10 pH/mV meter using an Accumet double junction semi-micro pH probe. The titration was carried out at room temperature (20 ± 2 °C).

Data were fit as for the alkaline transition using an equation analogous to eq 2 to extract the midpoint pH, pH_1/2, for acid unfolding. Plots of extinction coefficient versus pH were also fit to a pH dependent observed equilibrium constant, K_obs (pK_obs = pK_C(A/N)/[(1+10^n(pKa-pH)]), between the acid unfolded state, A, and the native state, N, using eq 3.

$${\text{ε}}_{\text{λcorr}}= \frac{\text{εA }+\text{ εN }\left[10\text{-pKC(A/N)}/1+10\text{n (pKa-pH)}\right]}{1+\left[10\text{-pKC(A/N)}/1+10\text{n(pKa-pH)}\right]}$$ (3)

In eq 3, ε_λcorr is the corrected extinction coefficient at the wavelength being monitored (ε_λ – ε₇₅₀), ε_A is the corrected extinction coefficient of the acid state and ε_N is the corrected extinction coefficient of the native state at that wavelength, pK_a is the acid dissociation constant of the group which protonates upon acid unfolding and pK_C(A/N) (= −logK_C(A/N)) is the conformational equilibrium constant at high pH for formation of the native state where the ionizable group is fully deprotonated. Given the pH range of acid unfolding, we assume pK_a is 4, corresponding to ionization of a carboxylate group such as a heme propionate.³⁸

pH Jump Stopped-flow Kinetics of the Alkaline Transition.

As previously reported,^39–40 pH jump stopped-flow experiments were executed at 25 °C using an Applied Photophysics SX20 stopped-flow spectrometer. A total of 5000 data points were collected on a logarithmic time scale monitoring at 398 nm. Data were collected on a 100 ms to a 100 s time scale depending on final pH. Both upward and downward pH jump data were collected in increments of 0.25 pH units. Initial sample conditions for upward pH jumps were 20 μM A51V Hu Cytc in 0.1 M NaCl (pH 6), which was mixed in a 1:1 ratio with 50 mM buffer of the desired pH (pH 7.5 – 11) in 0.1 M NaCl producing a 10 μM protein solution in 25 mM buffer in 0.1 M NaCl. Downward pH jumps were carried out in a similar manner beginning at pH 10.5 and jumping to the pH regime 7 – 8. Effluent was collected, and the final pH was measured with a Denver Instrument UB-10 pH/mV meter using an Accumet double junction semi-micro pH probe. The pH of the effluent was within error of the pH of the buffer used for mixing in all cases. Buffers were as follows: MES (pH 6.0−6.5), NaH₂PO₄ (pH 6.75−7.5), Tris (pH 7.75−8.75), H₃BO₃ (pH 9−10), and CAPS (pH 10–11). A minimum of 5 trials were collected at each pH. Data were fit to the appropriate exponential function using SigmaPlot v. 13.

Crystallization and Structure Determination of Human A51V Cytc.

A51V Hu Cytc was oxidized with K₃[Fe(CN)₆], followed by separation from the oxidizing agent and exchange into 50 mM sodium phosphate pH 7 using Sephadex G-25 chromatography. It was then concentrated to ~13.5 mg/mL using centrifuge ultrafiltration (Amicon Ultra-4 10,000 MWCO). Screening for crystallization conditions was carried out using the JCSG screening kits. The ratio of protein to reservoir solution in the drops in the 96 well sitting drop screening plates was 1:1. We obtained crystals from JCSG core II, well D6 (0.1 M sodium citrate pH 5.5, 40% w/v PEG 600). Additional vapor diffusion crystallization experiments were set up in a 24-well plate by expanding upon the pH and precipitant concentration of this initial condition. After 3 days to 2 weeks of equilibration at 20 °C, crystals diffracting to 1.82 Å in the C222₁ space group were obtained from a drop containing 1 μL of protein and 1 μL of 40% w/v PEG 600, 0.1 M sodium citrate pH 5.7. Crystals were cryoprotected with 20% glycerol and flash frozen in liquid N₂ for data collection.

The X-ray diffraction dataset was collected under cryogenic conditions at 100 K using the SMB beamline 9–2 of the Stanford Synchrotron Radiation Lightsource (SSRL) and a Pilatus 6 M detector. Diffraction data were processed using iMOSFLM⁴¹ and scaled and merged using Aimless.⁴² The initial electron density map was determined using phases obtained with the molecular replacement method using MOLREP,⁴³ incorporated in the CCP4i2 software suite,⁴⁴ and the coordinates of the K72A human Cytc structure (PDB ID: 5TY3)⁴⁵ as a search model. The initial model was built into a continuous electron density map and subsequently refined using REFMAC⁴⁶ and PHENIX.⁴⁷ The structure model was further refined to 1.82 Å resolution by multiple rounds of manual model rebuilding with COOT⁴⁸ and restrained refinement with REFMAC and PHENIX using 5 % of reflections for calculation of R_free. Data collection and refinement statistics of the final model are summarized in Table 1.

Table 1.

X-ray Crystallography and Data Collection and Refinement Statistics

PDB code	6DUJ
Beamline	SSRL SMB 9–2
Wavelength (Å)	1.07
Resolution range (Å)	33.32–1.82 (1.89–1.82)*
Space group	C 2 2 21
Unit cell dimensions
a, b, c (Å)	62.4, 184.4, 35.7
α, β, γ (°)	90, 90, 90
Unique reflections	18770 (1878)*
Multiplicity	6.5 (5.3)*
Completeness (%)	98.7 (99)*
Mean I/σ(I)	24.5 (6.1)*
Wilson B-factor	21.98
Rsym†	0.07 (0.27)*
Refinement
Rwork§	0.184 (0.292)*
Rfree§	0.235 (0.317)*
Number of total atoms	1787
protein	1610
heme	86
solvent	91
Total protein residues	210
RMS (bonds, Å)	0.02
RMS (angles, °)	1.62
Ramachandran favored (%)††	98
Rotamer outliers (%)††	0
Average B-factor (Å2)	32.34
macromolecules (Å2)	33.00
heme (Å2)	13.90
solvent (Å2)	38.80

^*Data for highest resolution shell are given in brackets.

^†R_sym =∑_hkl ∑_i | I_i (hkl)- 〈I (hkl)〉|/ ∑_hkl ∑_i I_i (hkl) where I_i (hkl) is the ith observation of the intensity of the reflection hkl.

^§R_work =∑_hkl || F_obs|-|F_calc||/ ∑_hkl |F_obs|, where F_obs and F_calc are the observed and calculated structure-factor amplitudes for each reflection hkl. R_free was calculated with 5% of the diffraction data that were selected randomly and excluded from refinement.

^††Calculated using MolProbity.⁵²

Guaiacol Assay of Peroxidase Activity.

Peroxidase activity was measured with the colorimetric reagent, guaiacol, using previously reported conditions and procedures.^{40, 45} The reaction was monitored at 25 °C using an Applied Photophysics SX20 stopped-flow apparatus. The formation of tetraguaiacol from guaiacol and H₂O₂ in the presence of Cytc was monitored at 470 nm. Cytc (4 μM) in 50 mM buffer, 0.1 M NaCl was mixed in a 1:1 ratio with guaiacol at four-fold the desired final guaiacol concentration in 50 mM buffer, 0.1 M NaCl to produce Cytc/guaiacol stock solutions in 50 mM buffer, 0.1 M NaCl. Each Cytc/guaiacol stock solution was mixed 1:1 with 100 mM H₂O₂ in 50 mM buffer, 0.1 M NaCl by the stopped-flow instrument yielding a final solution containing 1 μM Cytc, 50 mM H₂O₂ and guaiacol at the desired concentration in 50 mM buffer, 0.1 M NaCl. Concentration was determined using the extinction coefficients of H₂O₂ (ε₂₄₀ = 41.5 M⁻¹ cm⁻¹; average of published values^49–50) and guaiacol (ε₂₇₄ = 2,150 M⁻¹ cm⁻¹).⁵¹ Buffers used for peroxidase experiments were the same as those used in pH-Jump experiments. Final concentrations of guaiacol after mixing were 0, 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60, 80, and 100 μM.

The segment of the A₄₇₀ versus time data with the greatest slope following the initial lag phase was used to obtain initial velocity, v, at each guaiacol concentration. The data were fit to a linear equation and the slope from five repeats was averaged. The slope (dA₄₇₀/dt) was divided by the extinction coefficient of tetraguaiacol at 470 nm (ε₄₇₀ = 26.6 mM⁻¹cm⁻¹)⁵³ and multiplied by 4 (4 guaiacol consumed per tetraguaiacol produced) to give the initial rate of guaiacol consumption, v. The initial rate, v, was divided by Cytc concentration, plotted against guaiacol concentration and fit (SigmaPlot v. 13) to the Michaelis-Menten equation, eq 4, to obtain K_m and k_cat values.

$$\frac{v}{\left[\text{Cyt}c\right]}= \frac{k_{\text{cat}}\cdot \left[\text{Guaiacol}\right]}{K_{\text{m}}+\left[\text{Guaiacol}\right]}$$ (4)

RESULTS

Structure of A51V Hu Cytc.

A51V Hu Cytc crystallized in acetate buffer at pH 5.8. The crystals diffracted to 1.82 Å in the C222₁ space group with two Cytc molecules in the asymmetric unit. The overall fold of A51V Cytc (PDB entry 6DUJ: R_work and R_free values of 0.18 and 0.24, respectively) is similar to that of WT Hu Cytc (PDB entry 3ZCF) with an all-atom root-mean-square-deviation (RMSD) of 0.835 Å (using molecule A of each structure) between the two X-ray structures (Figure 1). The all-atom RMSD between the two molecules in the asymmetric unit of the A51V Hu Cytc structure is slightly higher (1.104 Å). The X-ray structures of the other THC4 variants of Hu Cytc, G41S²² and Y48H,²⁰ are also very similar to the WT X-ray structure.

Figure 1.

Overlay of the A51V (PDB ID: 6DUJ, chain A, dark gray) and WT (PBD ID: 3ZCF, chain A, light gray) Hu Cytc structures. Ω-loop C (residues 40 – 57) is shown in wheat (WT) and light purple (A51V). Ω-loop D (residues 70 – 85) is shown in light pink (A51V) and salmon (WT). Stick models are used for the heme and selected side chains. Buried water molecules are shown as red spheres. Hydrogen bonds are shown as yellow dashed lines.

There are small, localized structural changes. In particular, the larger steric size of valine versus alanine pushes the main chain of Ω-loop D away from Ω-loop C. The largest main chain shift is at Gly77 (Figure 1). This shift propagates across the loop to Ile81. There is also a change in the ring pucker of Pro76. However, the hydrogen bond (H-bond) network that couples Met80 to two buried waters and heme propionates 6 and 7 (HP6 and HP7) through the side chains of Tyr67, Thr78, Trp59, Asn52, Thr49, Tyr48 and Arg38 is largely unchanged in the A51V variant relative to that of the WT protein (Figure 1). The slight shift of the main chain of Ω-loop D around Gly77 does allow invasion of water between Ω-loops C and D (Figure 2). For the A51V structure, the carbonyl oxygen of Val51 is linked with the carbonyl oxygen of Pro76 through a network of three water molecules that are not observed in the WT structure (Figure 2). The same is observed for the other molecule in the asymmetric unit of the A51V variant (Figure S1).

Figure 2.

Hydrogen bond network (yellow dashed lines) across Ω-loop D in (a) A51V (chain A) and (b) WT (chain A) Hu Cytc. Coloring of Ω-loops C and D is as in Fig. 1. Ω-loop D is shown with stick models. Waters are shown as red spheres.

A closer examination of Ω-loop D shows that the cross-loop network of direct and water mediated H-bonds is much more sparse for the A51V variant versus that observed for WT Hu Cytc (Figure 2). Both WT and A51V Hu Cytc share main chain to main chain L68 to I85 and I75 to T78 H-bonds. Both also have a water mediated cross-loop H-bond network involving Asn 70, Lys72 and the carbonyl of Phe82. Water-mediated H-bonds are also largely retained on the periphery of the loop for the A51V variant as observed for the WT protein. However, the extensive network of water mediated H-bonds connecting the side chain of Lys72, and the main chain atoms of Phe82, Met80, Thr78 and Gly77 observed with the WT protein is missing in the A51V variant. The cross-loop H-bond network is even more sparse for the other molecule of A51V Hu Cytc in the asymmetric unit (Figure S1, chain C).

This observation suggests that Ω-loop D may be more dynamic for the A51V variant than for WT Hu Cytc. Crystallographic B-factors can provide a qualitative estimate of differential dynamics for variants of the same protein. Based on the difference in resolution alone (WT, 1.65 Å; A51V, 1.82 Å) a 20% increase in the crystallographic B-factors would be expected for the A51V variant.⁵⁴ For WT Hu Cytc, the average backbone B-factor is 18.4 ± 3.1 Ų compared to 27.6 ± 5.5 Ų for that of the A51V variant, a 50% increase. The largest increase in B-factors occurs in the C-terminal half of the protein following the site of the A51V substitution, with consistently higher B-factors in Ω-loop D (Figure S2). The large standard deviation of the B-factors in this region may reflect differences in intermolecular contacts between the two molecules in the asymmetric unit. Thus, the upper limit of the error bars in Figure S2 may be more reflective of the differences in dynamics in Ω-loop D for WT versus A51V Hu Cytc.

Our previous structural study of a K72A variant of yeast iso-1-Cytc (PDB entry 4MU8) shows that Arg38 can act as a gate that mediates access to a buried water channel.¹⁰ A similar effect has been also observed in the structure of G41S Hu Cytc (PDB entry 3NWV).²² The movement of Arg38 away from the heme allows access of water (and possibly H₂O₂) to the heme. Both proteins exhibit enhanced peroxidase activity. However, the Arg38 side chain in the A51V structure is in the same position as in the WT structure (i.e., no heme access) and that of the Y48H variant of Hu Cytc.²⁰

Global Stability of A51V Hu Cytc.

Global unfolding thermodynamics of A51V Hu Cytc was monitored by CD at 25 °C and pH 7.5 with the use of GdnHCl as denaturant. Figure 3 shows a plot of θ_222corr versus GdnHCl concentration for the A51V variant compared to WT Hu Cytc. Thermodynamic parameters from fits to a two-state model are given in Table 2. ΔG_u^°’(H₂O) of the A51V variant is lower than that of the WT protein by ~1.3 kcal mol⁻¹. The data for the G41S and Y48H variants were acquired at 15 °C²⁰ versus 25 °C for the A51V variant (Table 2). ΔG_u^°’(H₂O) for WT Hu Cytc is ~1.2 kcal/mol larger at 15 °C than at 25 °C (Table 2). If the temperature dependence of ΔG_u^°’(H₂O) is assumed to be similar for the variants, then ΔG_u^°’(H₂O) of A51V is larger than those for the other two naturally occurring variants linked to THC4, G41S and Y48H, by ~2.6 kcal mol⁻¹ and ~4.3 kcal mol⁻¹, respectively. Within error, the midpoint GdnHCl concentration for unfolding, C_m, is unchanged for the A51V variant relative to that of WT Hu Cytc, whereas C_m of the G41S and Y48H variants are decreased by ~0.4 M and ~0.6 M, respectively, relative to that of WT Hu Cytc.

Figure 3.

GdnHCl denaturation of A51V and WT Hu Cytc at 25 °C and pH 7.5. Corrected ellipticity at 222 nm, θ_222corr, is plotted versus GdnHCl concentration. Solid curves are fits to eq 1 in Experimental Procedures. Data shown with open circles were not used in the fits to the two-state model. Parameters obtained from the fits are given in Table 1. The data for WT Hu Cytc are from ref.²⁹ as reanalyzed in ref⁴⁵.

Table 2.

Thermodynamic Parameters for Global Unfolding of Hu Cytc Variants

Variant	T, °C	ΔGuo’(H2O), kcal mol−1	m, kcal mol−1 M−1	Cm, M
A51Va	25	8.2 ± 0.2	3.0 ± 0.1	2.7 ± 0.2
WT	25	9.46 ± 0.11b	3.71 ± 0.05b	2.55 ± 0.01b
	15	10.65 ± 0.55c	4.10 ± 0.25c	2.60 ± 0.05c
G41Sc	15	6.75 ± 0.10	3.0 ± 0.1	2.25 ± 0.05
Y48Hc	15	5.09 ± 0.16	2.57 ± 0.1	1.98 ± 0.05

^aData obtained at 25 °C in 20 mM Tris, pH 7.5, 40 mM NaCl.

^bParameters are from ref⁴⁵ for data obtained under the same conditions as the A51V variant.

^cParameters are from ref²⁰ for data obtained at 15 °C in 20 mM potassium phosphate pH 6.5, 50 mM KF.

In Figure 3, the unfolding transition appears to be broader for the A51V variant than for that of WT Hu Cytc. Consistent with this observation, fits of the A51V data to a two-state model yield a decrease of about 0.7 kcal mol⁻¹ M⁻¹ for the GdnHCl m-value compared to that of WT Hu Cytc (Table 2). The m-values of the A51V and G41S variants are the same and about 0.4 kcal mol⁻¹ M⁻¹ larger than that of the Y48H variant. Thus, a common feature of the variants linked to THC4 is a loss in the cooperativity of the folding of Hu Cytc.

Local Stability Assessed by the Alkaline Conformational Transition.

Cytc undergoes a conformational change at moderately alkaline pH.^55–56 In the terminology of Theorell and Åkesson,⁵⁷ this conformational change is the conversion from state III (native state) to state IV (alkaline state) and involves replacement of the Met80 heme ligand with either Lys73 or 79.^{37, 58} For mammalian cytochromes c, the apparent pK_a of this transition is typically between 9 and 9.5.⁵⁹ Above pH 12, state V forms with replacement of the lysine ligand with hydroxide in mammalian cytochromes c.^{57, 60} However, the less stable yeast iso-1-cytochrome c forms states IV and V at lower pH.^{37, 61} The alkaline transition correlates well with the local stability of Ω-loop D.^{33, 58} The stability of Cytc with respect to the alkaline conformational transition and the accessibility of states which promote peroxidase activity often correlate well.^{18, 40} Thus, the local unfolding thermodynamics for the alkaline conformational transition of A51V Hu Cytc was determined by pH titration, monitored at 695 nm to follow the loss of Met80-heme iron ligation⁸ when a lysine (Lys72, Lys73 or Lys79) from Ω-loop D binds to the heme. Comparison of data for the A51V variant to previously published data²⁹ for WT Hu Cytc (Figure 4) show that the alkaline transition is shifted to significantly lower pH. The fits of the data for A51V to eq 2 (Experimental Methods) show that the number of protons, n, linked to the conformational change is approximately equal to 1 (Table 3), consistent with a one proton process as normally observed for the alkaline transition of Cytc.^55–56 The A51V substitution causes a decrease in the midpoint pH, pH_1/2, of the alkaline transition of about 1 unit relative to WT Hu Cytc. Within error, the pH_1/2 of the A51V variant is similar to those of the G41S and Y48H variants, obtained by Worrall and coworkers also using data at 695 nm (Table 3).²⁰ However, the pH_1/2 reported by Bren, Ledgerwood and coworkers for the G41S variant using data at 695 nm is somewhat lower (Table 3).²¹ It should be noted that the alkaline transition is sensitive to buffer conditions,⁵⁹ which vary for these different measurements.

Figure 4.

Plots of ε_695corr versus pH for the alkaline transition of the A51V variant versus WT Hu Cytc.²⁹ Data were collected at room temperature (22 ± 2 °C) in 0.1 M NaCl solution. Solid lines are fits to eq 2 in Experimental Methods.

Table 3.

Thermodynamic Parameters for the Alkaline Transition of WT and THC4-linked Variants of Hu Cytc

Variant	pH1/2	n
A51Va	8.56 ± 0.04	0.9 ± 0.1
WT	9.54 ± 0.03b	1.03 ± 0.02b
	9.3 ± 0.2c	-d
G41S	8.5 ± 0.2c	-d
	7.8 ± 0.3e	-d
Y48H	8.4 ± 0.1c	-d

^aData were collected at 22 ± 2 °C in 0.1 M NaCl.

^bParameters are from ref.²⁹ and were collected at 22 ± 2 °C in 0.1 M NaCl.

^cParameters are from ref²⁰ for data collected at 695 nm and room temperature in 20 mM sodium phosphate.

^dData were fit assuming a one proton process.

^eParameters are from ref²¹ for data collected at 695 nm and 25 °C in the 50 mM sodium phosphate.

Kinetics of the Alkaline Conformational Transition of A51V Hu Cytc.

In order to determine the actual rates of interconversion between the native and alkaline conformations, stopped-flow pH jump methods were employed. Both upward and downward pH jumps were monitored at 398 nm to follow heme-ligand changes linked to rearrangement of Ω-loop D. Because there are three lysines, Lys72, Lys73, and Lys79, present in Ω-loop D, as many as three kinetic phases are possible. In upward pH jump experiments, the A51V variant shows two slow phases from pH 7.5 to 10 (k_obs,1, A_1u) or 10.5 (k_obs,2, A_2u) and a fast phase (k_obs,3, A_3u) from pH 8.5 to 11 (Figures S3, 5 and 6). Previous studies on the G41S and Y48H variants linked to THC4 also observed deviation from single-exponential behavior for the slow phase.⁶² However, the deviation was considered small so the slow phase was fit as a single first-order kinetic process. The study of the G41S and Y48H variants did not acquire downward pH jump data. We observe two slow phases in downward pH jump data, as well. Therefore, we have elected to fit the entire data set assuming the presence of two slow phases.

Figure 5.

Rates constants for the alkaline transition of the A51V variant of Hu Cytc at 25 °C in 0.1 NaCl. Plots of (a) k_obs,1, (b) k_obs,2, and (c) k_obs,3 versus pH from upward (filled symbols) and downward (open symbols) pH jump experiments. Solid lines are fits of the combined upward and downward k_obs versus pH data to eq 5 in the Discussion.

Figure 6.

Plots of amplitude versus pH for the three kinetic phases observed for the alkaline conformational transition of A51V Hu Cytc. Color scheme is the same as for Figure 5. Filled symbols are used for upward pH jumps (A_1u, A_2u and A_3u). Open symbols are used for downward pH jumps from pH 10.5 (A_1d, A_2d and A_3d). Data points are the average and standard deviation of a minimum of five trials. A_1u versus pH and A_2u versus pH data were fit to eq 6 in the Discussion (solid lines). In these fits, k_b and k_f in eq 6 were set equal to the values determined from the fits of k_obs versus pH to eq 5 in Figure 5. Data points connected with dashed lines were not included in these fits. A_3u versus pH data were fit to Henderson-Hasselbalch equation (solid line).

The rate constant for one of the two slow phases, k_obs,1 remains relatively constant below pH 8.25, with values near 0.04 s⁻¹ (Table S2). Above pH 8.25, k_obs,1 increases to ~0.35 s⁻¹ as pH approaches 10 (Table S2 and Figure 5). The amplitude for this phase A_1u increases from around 0.007 at pH 7.5 to 0.057 at pH 8.75, and then begins to decrease above pH 8.75 (Table S2 and Figure 6) as the other slow phase (k_obs,2, A_2u) becomes the dominant phase.

The rate constant for the other slow phase, k_obs,2, also is relatively constant below pH 8.25, with values near 0.09 s⁻¹ (Table S2 and Figure 5). Above pH 8.25, k_obs,2 increases to 3.6 s⁻¹ as pH approaches 10.5. The amplitude for this phase, A_2u, increases from around 0.005 at pH 7.5 to 0.09 at pH 9.5, and then decreases above pH 9.5 (Table S2 and Figure 6).

At pH 8.5 and above a fast phase (k_obs,3, A₃, Table S2) grows in. The rate constant for this fast phase, k_obs,3, cannot be obtained with high precision between pH 8.5 and 9.5. Values near 23 s⁻¹ begin increasing above pH 8.5, approach 50 s⁻¹ at pH 9.5, and remain around 30 s⁻¹ above pH 10. The amplitude for the fast phase, A_3u, is small initially, but grows rapidly above pH 9.25, becoming the dominant phase as the two slow phases disappear above pH 10 (k_obs,1, A_1u) and pH 10.5 (k_obs,2, A_2u).

To obtain accurate rate constants in the lower pH regime, downward pH jump experiments were carried out beginning at pH 10.5 and jumping to pH 7–8 (Figure 5, Table S3). Downward pH jump data were fit to a triple exponential equation (Figure S4). The observed rate constants for the two slow phases in the downward pH jumps correspond well to k_obs,1 and k_obs,2, observed in upward pH jumps (Figures 5A and 5B). The observed rate constants for the fast phase in the downward pH jumps corresponds well to k_obs,3 (Figure 5C).

Acid Unfolding of A51V and WT Hu Cytc.

The pH of intermembrane space of mitochondria is more acidic (pH = 6.88 ± 0.08) than the matrix (7.8 ± 0.2) or the cytosol (7.59 ± 0.01).⁶³ Thus, it is important to determine the stability of A51V Hu Cytc with respect to pH to evaluate the role of Ω-loop C with regard to the accessibility of alternate conformers with the high spin heme believed to be necessary for peroxidase activity. The unfolding thermodynamics for the acid unfolding of A51V Hu Cytc was determined by pH titration. Figure 7 shows plots of the corrected extinction coefficients at 695 nm (Met80 ligation), ε_695corr, and 622 nm (high spin heme), ε_622corr, versus pH for the acid unfolding of A51V compared to previously reported data for WT Hu Cytc.⁶⁴ The plots show that the A51V variant is significantly less stable to acid unfolding than WT Hu Cytc. For both proteins, the data monitored at 622 nm yield a broader transition than the data at 695 nm. The somewhat different behaviors of the 695 nm data and the 622 nm data are a result of the complexity of acid unfolding. Acid unfolding leads to sequential formation of states II and I from the native state (state III). While state II is compact, state I is disordered.⁶⁵ High ionic strength stabilizes state II with respect to state I, but under the low ionic strength conditions of our experiments, the two transitions are not well-separated.^{38, 65} State II is a mix of low spin and high spin (Met 80 replaced by H₂O) states.^{38, 66} Originally, the low spin state was thought to involve Met80 ligation,³⁸ however, more recent resonance Raman data suggest that ligation by a histidine (likely His 33) is involved in the low spin state II conformer.⁶⁶ The loss of the 695 nm band can be due to both formation of another low spin species and a high spin state and so prefect overlap of acid unfolding monitored at 695 nm and 622 nm (high spin) is not expected.

Figure 7.

Plots of ε_695corr and ε_622corr versus pH for acid unfolding of WT and A51V Cytc. Data were acquired at 22 ± 2 °C in the presence of 100 mM NaCl. The solid curves are fits to Henderson-Hasselbalch equation. Data for the WT protein are from ref.⁶⁴.

Fits of the data to the Henderson-Hasselbalch equation show that the midpoint pH, pH_1/2, for acid unfolding shifts ~0.7 unit to higher pH for the A51V variant relative to that of WT Hu Cytc (Table 4). A modest decrease in pH_1/2 (although within error for the A51V variant) is also observed for data monitored at 622 nm relative to 695 nm (Table 4). The number of protons, n, linked to acid unfolding for data monitored at 622 nm versus 695 nm also decreases (Table 4). Consistent with these observations, an isosbestic point near 647 nm is lost at pH values at or just below pH_1/2 for acid unfolding for both proteins (Figure S5). Loss of this isosbestic point may signal the onset of the overlapping state II to state I transition of acid unfolding.^{38, 65} Data were also fit to eq 3 in Experimental Methods to provide an equilibrium constant for the acid to native state transition (pK_C(A/N), Table 4).

Table 4.

Thermodynamic Parameters for the Acidic Transition of WT and A51V Hu Cytc at 22 ± 2 °C

	695 nm			622 nm
Variant	pH1/2	pKC(A/N)a	n	pH1/2	pKC(A/N)a	n
A51V	3.19 ± 0.09	−1.4 ± 0.3	1.8 ± 0.4	3.13 ± 0.06	−1.1 ± 0.2	1.3 ± 0.1
WTb	2.50 ± 0.03	−2.50 ± 0.07	1.64 ± 0.07	2.41 ± 0.03	−1.73 ± 0.06	1.13 ± 0.08

^aAssumes the pK_a of the ionizable group(s) is 4.

^bParameters are taken from ref⁶⁴.

Peroxidase Activity of A51V Hu Cytc.

The peroxidase activity of A51V Hu Cytc was measured by monitoring the formation of tetraguaiacol from guaiacol in the presence of H₂O₂. Michaelis-Menten plots with respect to guaiacol concentration were generated to permit extraction of k_cat and K_m across the pH range 5 to 8 (Figure 8a). The A51V variant shows a significant increase in k_cat values relative to those of WT Hu Cytc (Figure 8b). The increase in k_cat values for the A51V variant relative to those of WT Hu Cytc is more pronounced as pH decreases. k_cat increases ~7-fold at pH 6 versus ~14-fold at pH 5 for k_cat of the A51V variant versus k_cat of WT Hu Cytc (Table 5). Relative to the K_m values of the WT protein, those of A51V Cytc are not strongly affected (Table 5).

Figure. 8.

Peroxidase activity of WT and A51V Hu Cytc. (a) Michaelis-Menten plots for the A51V variant of Hu Cytc at pH values from 5 to 8. The solid curves are fits to eq 4 (Experimental Methods). Data were acquired at 25 °C in 50 mM buffer. (b) k_cat versus pH for WT (previously published,^{45, 64} dark green bars) and A51V (red bars) Hu Cytc. Error bars are the standard deviation from three independent experiments.

Table 5.

Michaelis-Menten Parameters as a Function of pH at 25 °C for the Peroxidase Activity of WT and A51V Hu Cytc

pH	Km, μM		kcat, s−1
	WTa	A51I	WTa	A51V
5.0	32 ± 2	22 ± 3	0.11 ±0.03	1.6 ± 0.1
6.0	14 ± 2	10 ± 2	0.108 ± 0.007	0.798 ± 0.006
7.0	5.7 ± 0.3	11 ± 2	0.112 ± 0.002	0.689 ± 0.005
8.0	11 ± 2	10 ± 1	0.06 ± 0.01	0.44 ± 0.01

^aParameters from references^{45, 64}

DISCUSSION

Effect of the A51V Substitution on the Global and Local Stability and Structure of Hu Cytc.

Consistent with previous work performed on two other naturally occurring variants, G41S and Y48H,²⁰ the A51V variant of Hu Cytc shows both decreased global stability and decreased stability of the native state relative to subglobal unfolding of Ω-loop D to form the alkaline state (Tables 2 and 3). The thermodynamic properties of the foldons of Cytc are consistent with sequential unfolding.⁶⁷ In particular, mutations that stabilize or destabilize a foldon affect the stability of more stable foldons that unfold after the mutated foldon by the same amount as the mutated foldon, whereas the stabilities of less stable foldons that unfold before the mutated foldon are unaffected.^68–70 A good correlation between mutation-induced changes in the stability of Ω-loop D as monitored by the alkaline transition and the effect of these mutations on global stability has been observed.^{33, 58} The A51V substitution leads to a decrease in the pH_1/2 of the alkaline transition of about 1 unit, similar to the decreases in pH_1/2 observed for the other THC4-linked variants of Hu Cytc (Table 3). A decrease in pH_1/2 by 1 unit corresponds to ~1.3 kcal/mol (ΔΔG_alk = 2.3RTΔpH_1/2) very close to the change in global stability for the A51V variant versus WT Hu Cytc (Table 2) and consistent with sequential unfolding of the foldons of Cytc. We previously obtained similar correspondence between changes in global stability and changes in the stability of Ω-loop D as determined via the alkaline transition for I52A and I52T variants of yeast iso-1-Cytc.³³ Comparing the data in Tables 2 and 3, there is obviously a poor correlation between local and global stability for the other two THC-linked variants of Hu Cytc.²⁰ This observation likely results from the incomplete coupling between the stabilities of the two least stable foldons, the Ω-loop C foldon and the Ω-loop D foldon, that has been noted in studies on horse Cytc.⁶⁸ For example, it was shown that destabilization of Ω-loop C of horse Cytc by a T47G mutation within the loop or by decreasing pH destabilizes all more stable foldons by the same amount, except for the Ω-loop D foldon, which is destabilized by about half the amount of the other foldons. These results indicate that unfolding of the Ω-loop D foldon sometimes occurs independently of the unfolding of the Ω-loop C foldon and sometimes occurs after unfolding of the Ω-loop C foldon such that stability changes in Ω-loop C may only be partially communicated to Ω-loop D, while fully affecting the global stability of the protein. Therefore, the relative effect of an Ω-loop C mutation on global stability versus the stability of the alkaline state relative to the native state will depend on how the particular mutation affects the proportion of independent versus sequential unfolding of the Ω-loop C and Ω-loop D foldons.

Previous studies showed a significant loss in the cooperativity of unfolding for THC4-linked Hu Cytc variants as measured by the magnitude of the denaturant m-value. Both the A51V and G41S substitutions cause a 20% decrease in the GdnHCl m-value whereas the Y48H variant causes a 30% decrease in the GdnHCl m-value relative to that of WT Hu Cytc (Table 2). Because GdnHCl unfolding monitored by circular dichroism at 222 nm (α-helix) is not strongly sensitive to unfolding of Ω-loops, the smaller m-values could reflect partial unfolding of Ω-loop C (and possibly Ω-loop D) prior to global unfolding (loss of helical structure). This type of behavior has been observed for histidine variants (K73H and K79H) in Ω-loop D of yeast iso-1-Cytc.^{30, 39, 71–74} Loss of structure in Ω-loop C prior to the major unfolding transition monitored by CD would indicate destabilization of this substructure by the A51V, G41S and Y48H substitutions. We note that decreased m-values can also reflect a more compact denatured state.⁷⁵

The loss of ordered water structure around Ω-loop D of the A51V variant (Figure 2) and the overall increase in the magnitude of crystallographic B-factors for the A51V variant are (Figure S2) consistent with destabilization of Ω-loops C and D. H/D exchange experiments on the G41S and Y48H variants also support this interpretation.^19–20

The nature of out-of-plane distortion of the heme cofactor appears to be conserved within different classes of heme proteins.⁷⁶ Ruffling is predominant in c-type cytochromes,^76–77 whereas peroxidases exhibit strong saddling and moderate ruffling.⁷⁶ Ruffling affects the electronic structure of c-type cytochromes and has been shown to be correlated to redox potential.^77–78 X-ray structures of WT,¹⁸ G41S²² and Y48H²⁰ human Cytc along with the A51V structure presented here allow the effect of the substitutions in all three THC-linked variants on heme out-of-plane distortion, D_oop, to be compared. Using the NSD program^79–80 to analyze D_oop of the heme and its normal mode components, we find that the magnitude of D_oop of the heme ranges from 0.6 – 1 Å and follows the order: G41S > WT ≈ Y48H > A51V (Table S4). In all cases, ruffling (B_1u component) is the predominate form of distortion as expected for c-type cytochromes,⁷⁷ following the order: G41S > WT > Y48H > A51V (Table S4). Saddling (B_2u component), while a much smaller component of D_oop, does contributes significantly to heme distortion. Relative to WT human Cytc, saddling increases for the Y48H and G41S variants and decreases for the A51V variant (Table S4). Thus, the A51V substitution causes a significant decrease in D_opp, primarily due to a decrease in ruffling, but with a notable contribution from a decrease in saddling. However, overall there no clear trend for the effect of the THC-linked mutations on heme distortion, suggesting that it may not be the predominate factor in the relationship to disease or the enhanced peroxidase activity observed for these variants.

Effect of the A51V Substitution on the Slow Phases of the Alkaline Transition.

The kinetic data for the alkaline transition of A51V Cytc follows the standard mechanism, a rapid deprotonation equilibrium (K_H or pK_H), which triggers a conformational rearrangement in which a lysine replaces the Met80 heme ligand. For this mechanism,⁸¹ the observed rate constant, k_obs, increases with pH and then levels off (Figure 5, eq 5).

$$k_{\text{obs}}=k_{\text{b}}+k_{\text{f}}\left(\frac{1}{1+{10}^{\text{p}{K}_{\text{H}}-\text{pH}}}\right)$$ (5)

The forward rate constant, k_f, corresponds to formation of the alkaline state from the native state following the triggering deprotonation, and the backward rate constant, k_b, is for the return to the native conformer from the alkaline state. Eq 6 describes the pH dependence of the amplitude for this mechanism,⁸¹ where ΔA_t is the total amplitude when the alkaline transition goes to

$$\mathrm{\Delta }\text{A}=\mathrm{\Delta }{\text{A}}_{\text{t}}\left(\frac{1}{1+\frac{k_{\text{b}}}{k_{\text{f}}}\left(1+{10}^{\text{p}{K}_{\text{H}}-\text{pH}}\right)}\right)$$ (6)

completion. The other parameters are as defined for eq 5. In good agreement with this kinetic model, both k_obs,1 and k_obs,2 increase as pH increases. Parameters for the fits of the k_obs,1 and k_obs,2 versus pH to eq 5 (Figure 5) are given in Table 6. pK_H can also be evaluated with eq 6 from the plots of A_1u and A_2u versus pH in Figure 6 using the k_f and k_b values obtained from the fits to eq 5 (Table 6). The pK_H values obtained from rate constant and amplitude (values in brackets in Table 6) data are within error the same for k_obs,1, indicating that k_f and k_b obtained from rate constant data are reliable. The values of k_f, k_b and pK_H from kinetic data can also be used to calculate the pH_1/2, pH_1/2^calc. For k_obs,1, pH_1/2^calc is within error the same as the pH_1/2 of 8.56 ± 0.04 obtained from equilibrium measurements.

Table 6.

Kinetic Parameters for the Alkaline Transition of A51V Hu Cytc at 25 °C

Variant	Rate constant	kf, s−1 a	kb, s−1 a	pKH a	pKC b	pH1/2calc c
A51V	kobs,1	0.32 ± 0.03	0.0368 ± 0.008	9.3 ± 0.1 (9.5 ± 0.3)d	−0.9 ± 0.1	8.4 ± 0.2 (8.6 ± 0.3)e
	kobs,2	6.2 ± 0.3	0.11 ± 0.02	10.37 ± 0.04 (10.9 ± 0.1)d	−1.75 ± 0.08	8.61 ± 0.09 (9.1 ± 0.1)e
I81Af	kobs,1	1.6 ± 0.2	0.011 ± 0.003	10.7 ± 0.2 (10.8 ± 0.1)d	−2.2 ± 0.1	8.6 ± 0.2 (8.7 ± 0.2)e
	kobs,2	3.8 ± 0.2	0.07 ± 0.01	10.8 ± 0.1	−1.74 ± 0.08	9.1 ± 0.1
V83Gf	kobs,1	0.7 ± 0.2	0.039 ± 0.003	11.2 ± 0.1	−1.2 ± 0.1	9.9 ± 0.2
	kobs,2	6 ± 1	0.13 ± 0.01	11.2 ± 0.1	−1.68 ± 0.08	9.6 ± 0.2

^aFrom fit of data in Fig. 5 to eq 5. Errors are the standard errors in the parameters from the fit to eq 5.

^bpK_C = −LogK_C, where K_C = k_f/k_b, and K_C is the conformational equilibrium constant after the triggering deprotonation. Error is from standard propagation of the errors in k_f and k_b.

^cpH_1/2^calc = pK_H – pK_C.

^dFrom fit of amplitude data in Fig. 6 to eq 6 using k_f and k_b in this table.

^eCalculated using pK_H from amplitude data.

^fParameters are from ref.⁶⁴.

For k_obs,2, k_f, k_b and pK_H obtained from rate constant versus pH data yield pH_1/2^calc that is also similar to the pH_1/2 from equilibrium data (Table 6). However, the pK_H obtained from amplitude versus pH data is considerably higher (as is the pH_1/2^calc, Table 6), suggesting that the parameters obtained from either the rate constant or amplitude data may be less reliable for the k_obs,2, A₂ slow phase. Constraining k_f in the fits of k_obs,2 versus pH to the range of 10 – 12 s⁻¹ returns values of k_b (0.16 – 0.17 s⁻¹) that are consistent with k_b obtained in downward pH jump experiments (Table S3) and yields values of pK_H from 10.7 – 10.8. Values of pK_H obtained from the amplitude data with these values of k_f and k_b remain near 10.9. While there may be some uncertainty in the value of pK_H for k_obs,2, its value is 1 – 1.5 units higher than pK_H for k_obs,1 allowing the alkaline conformer corresponding to the k_obs,1 phase to populate at lower pH despite a less favorable conformational equilibrium (by a factor of ~7, see pK_C in Table 6).

Recent work compared the alkaline transition kinetics of WT Hu Cytc and V83G and I81A variants in Ω-loop D to the naturally occurring Y48H and G41S variants in Ω-loop C.⁶² The value of pK_H was observed to decrease by about 1 unit for the Ω-loop C variants relative to WT Hu Cytc and the Ω-loop D variants. For WT and all variants, pK_C was unaffected with a value near −2.⁶² The absolute values of the kinetic parameters reported here cannot be compared directly because our data were fit to two slow phases rather than a single slow phase. However, we have recently reported kinetic data for the V83G and I81A variants of Hu Cytc, which we fit assuming two slow phases.⁶⁴ Values of pK_H for both the k_obs,1 and k_obs,2 phases were within the range of 10.7 – 11.2 for kinetic data fit with two slow phases for these variants (Table 6).⁶⁴ As for the G41S and Y48H variants, the increased access to alternate conformers for the A51V variant appears to result from a decrease in the pK_H of the triggering deprotonation. For the A51V variant, the decrease in pK_H appears to operate only on the alkaline state corresponding to k_obs,1 (Table 6) suggesting that the triggering deprotonation controlling the dynamics of different parts of Ω-loop D is not the same.

The I81A variant of Hu Cytc also shows enhanced kinetic accessibility of only one of the two kinetically distinguishable alkaline conformers in 0.1 M NaCl.⁶⁴ In this case, pK_H for k_obs,1 is unchanged whereas pK_C is more favorable because k_f is larger and k_b is smaller (Table 6). Notably, k_f and k_b derived from k_obs,1 for the V83G variant are similar to those of the A51V variant yielding a similar less favorable pK_C for these two variants (Table 6). For all three variants, the parameters (k_f, k_b, pK_H and pK_C) obtained for k_obs,2 are similar. These data indicate that the dynamics of Ω-loop D is bipartite with the conformational dynamics corresponding to k_obs,1 being more sensitive to mutation. Our studies of an A81I variant of yeast iso-1-cytochrome c were consistent with assignment of the alkaline conformer affected by this mutation to the one with Lys79 bound to the heme.⁴⁰ Thus, it is tempting to assign k_obs,1 to the Lys79-mediated alkaline transition. However, as noted in our report on the I81A and V83G variants,⁶⁴ additional mutagenesis studies would be required to confirm this assignment. In the case of the Ω-loop D variants, the stability of the part of Ω-loop D that mediates the dynamics corresponding to k_obs,1 is affected, whereas for Ω-loop C variants, it appears that the pK_H of the triggering ionization corresponding to k_obs,1 is affected.

Effect of the A51V Substitution on the Fast Phase of the Alkaline Transition.

Of equal interest, is the observation that the fast phase reported for mammalian Cytc near pH 10 and above^{45, 62, 64, 82–84} appears near pH 8.5 for A51V Hu Cytc. This phase is often attributed to a transient process involving weakening of the heme-Met80 bond or displacement of Met80 by hydroxide (which could be viewed as a transient form of state V).^83–84 EPR data for the transient formed by the Y48H variant at pH 11 show that it is a high spin species and thus relevant for peroxidase activity.⁶² The A51V variant appears to affect the fast phase differently than the G41S and Y48H variants. For the A51V variant, k_obs,3 decreases from pH 9 to 10.5 (Figure 5), consistent with deprotonation occurring after formation of the fast phase species as given by eq 7,^{62, 81–82} where k_obs decreases to the magnitude of k_f for pH > pK_H. This behavior also is

$$k_{\text{obs}}=k_{\text{f}}+k_{\text{b}}\left(\frac{1}{1+{10}^{\text{pH}-\text{p}{K}_{\text{H}}}}\right)$$ (7)

observed for the WT protein.^{62, 81–82} For both the G41S and Y48H variants, k_obs,3 increases with pH consistent with deprotonation preceding formation of the fast phase species (eq 5).⁶² In this regard, the A51V variant is less perturbative than the G41S and Y48H variants relative to WT Hu Cytc. For A51V Hu Cytc, k_obs,3 increases below pH 10 (pH 11 for WT) consistent with the pK_H for the ionization of the group controlling formation of the transient species being less than 10.

In the study on the G41S and Y48H variants,⁶² it was noted that the pK_a of His18 is expected to be near 11.⁸⁵ These authors suggested that redistribution of electron density into the His18 heme ligand when the Met80 heme ligand dissociates during the conformational change associated with formation of the transient could occur for WT Hu Cytc. This electron density redistribution could decrease the pK_a for deprotonation of the pyrrole-NH of His18 to near 9 causing release of a proton after the conformational change. This proposal also is consistent with our data for the A51V variant in Figure 5C. In the case of the Y48H and G41S variants,⁶² it was proposed that the greater dynamics of Ω-loop C provides direct access of bulk water to the pyrrole-NH of His18 and thus direct deprotonation of this group, with a pK_a near 11,^{62, 85} occurs before Met80 dissociation leading to kinetics consistent with eq 5. The higher global stability of the A51V variant, compared to the G41S and Y48H variants, would likely decrease the dynamics of Ω-loop C for the A51V variant relative to the G41S and Y48H variants preserving the mechanism for the fast phase observed for WT Hu Cytc.

For the A51V variant, the transient phase is also observed in downward pH jump experiments indicating that there is a measureable equilibrium population of a species that is normally a kinetic transient. For yeast iso-1-Cytc, the midpoint pH for formation of state IV is ~8.6^{37, 61} similar to that of A51V human Cytc. State V, which involves replacement of lysine-heme ligation with hydroxide, forms with a midpoint pH between 10.5 and 11 for the yeast protein.⁶¹ Given that our downward pH jump experiments start at pH 10.5, the presence of the fast phase in downward pH jumps may indicate equilibrium population of state V for the A51V variant at pH 10.5 and above.

Our data from downward pH jump experiments indicate that k_b is 15 – 20 s⁻¹ (Figure 5, Table S3). Based on eq 7, k_f is near 25 s⁻¹ (k_obs,3 near pH 10.5, Figure 5C, Table S2), yielding K_C ~ 1.5 for formation of the transient species. For the Y48H and G41S variants, k_f for formation of the transient high spin species is ~150 s⁻¹ and k_b is ~20 s⁻¹, yielding K_C ~ 7.5 for these variants.⁶² For WT, k_f ~ 20 s⁻¹ and k_b > 100 s⁻¹ indicating that K_C is < 0.2.⁶² Thus, the stability of the high spin transient follows the order WT < A51V < G41S ≈ Y48H. The amplitude, A_3u, of the fast phase as a function of pH for A51V Hu Cytc (Figure 6) yields a midpoint pH, pH_1/2, of 10.23 ± 0.03 when fit to the Henderson-Hasselbalch equation. For WT Hu Cytc, pH_1/2 is ~11 for population of the transient.⁶² Thus, the transient species populates more effectively and is available at lower pH for the Ω-loop C variants. These data suggest that this high spin species will be nearer in energy to the native state structure at pH values above 7 than it is for the WT protein. For enzymes, often alternate conformations that are nearby in energy to the native state drive catalysis.^86–87 Given that the fast phase corresponds to heme with an available coordination site,⁸³ the greater availability of this species might also enhance the peroxidase activity of the THC4-linked variants, as proposed previously.⁶²

Effect of the A51V Substitution on Acid Unfolding of Hu Cytc.

The acid unfolding of Cytc is a complex, multiproton process^{38, 65, 88–89} typically involving 2 – 3 protons.^{8, 73} However, cooperativity of acid unfolding is readily altered by amino acid substitutions.^{35, 39–40, 90} We have shown that a G83V substitution in iso-1-Cytc leads to biphasic acid unfolding which appears to be linked to increased peroxidase activity at mildly acidic pH relative to WT iso-1-Cytc.⁴⁰ For G83V iso-1-Cytc, the loss in cooperativity of acid unfolding appears to weaken the heme-Met80 bond at mildly acidic pH. Acid unfolding of WT and A51V Hu Cytc exhibit monophasic behavior (Figure 7). The pH_1/2 of the A51V variant is ~0.7 units higher than that of WT Hu Cytc. The decrease in stability of the native state toward acid unfolding to a high spin state (622 nm data) for the A51V variant relative to WT Hu Cytc is 1.8 ± 0.3 kcal/ mol (ΔΔG^°’_A51V/WT = 2.3RT[(pK_C(A/N)(WT) - pK_C(A/N)(A51V)] = −1.8 ± 0.3 kcal/mol).

The pH of intermembrane space of mitochondria is more acidic than the matrix or the cytosol,⁶³ and cytosolic pH decreases further to 5.8 during apoptosis.⁹¹ The greater accessibility of the high spin conformer for the A51V variant versus the WT protein, as indicated by the acid unfolding data in Table 4, demonstrates the importance of Ω-loop C for controlling access to conformers competent for peroxidase activity at biologically relevant pH values. The increase in k_cat for peroxidase activity for A51V versus that of WT Hu Cytc at pH 5 supports this contention (Figure 8). Even though the high spin conformer may not be populated at equilibrium at pH 5 (Figure 7), the A51V substitution brings the high spin state closer to the energy of the native state at pH 5, making it a more accessible alternate conformer that can drive catalysis.^86–87 Recent work shows that horse Cytc bound to CL vesicles populates conformers with the high spin heme necessary for peroxidase activity as pH decreases below 7.⁹² Thus, the effect of the A51V substitution might be more pronounced at acidic pH in the presence of CL-containing membranes, further indicating the potential importance of Ω-loop C for controlling access to peroxidase-competent conformers. We note that the broader transition observed at 622 versus 695 nm may indicate the presence of a low spin His-heme intermediate during acid unfolding,⁶⁶ which may somewhat lower access to high spin conformers below pH 7.

Effect of the A51V Substitution to Hu Cytc on Apoptotic Peroxidase Activity.

Previous studies on the peroxidase activity of the THC4-linked variants of Hu Cytc, G41S and Y48H, show that they induce 3- and 7-fold increases in peroxidase activity at pH 6.5 with 2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrate.^{20, 23, 62} These results are consistent with the magnitude of enhancement of peroxidase activity that we observe at pH 6 and 7 for the A51V variant with guaiacol as substrate. For the G41S variant,²³ peroxidase activity was measured over a broad pH range. Consistent with our results for the A51V variant (Figure 8, Table 5), a significant increase in peroxidase activity relative to WT Hu Cytc was observed as pH decreased from 7.5 to 5. Given the lower pH in the intermembrane space and in the cytosol during apoptosis noted above, these studies on the naturally occurring Ω-loop C variants highlight the importance of this loop in modulating the peroxidase activity of Cytc early in apoptosis.

The increase in peroxidase activity of A51V Hu Cytc relative to that of WT Hu Cytc is lowest at pH 7 (~6-fold) and somewhat higher at both pH 6 and 8 (~7-fold, Table 5), suggesting that different mechanisms might provide access to peroxidase competent conformers above and below pH 7. Increased peroxidase activity is often linked to greater accessibility of alkaline conformers,^{18–21, 40} although the correlation is not perfect.^{23, 62} All three THC4-linked variants show a similar increase in the accessibility of the alkaline state (pH_1/2 decreases by 1 unit, Table 3), which appears to be linked to a decrease in the pK_H for the triggering ionization but ~1 unit (Table 6).⁶² Based on the X-ray structures of the G41S^22–23 and Y48H⁶² variants, which both show disruptions to the hydrogen bond network around heme propionate 7 (HP7), a role for HP7 in this ionization was proposed.⁶² For the A51V variant, this network is less affected (Figure 1), but because of the increase in the backbone B-factors (Figure S2), the environment of HP7 may be affected for the A51V variant, too. A number of studies indicate that one of the heme propionates of mitochondrial Cytc has a pK_a >9, while the other has a pK_a < 4.5.^{8, 93–94} Initially, the pK_a < 4.5 was assigned to heme propionate 7 (HP7) because of the nearby positively-charged Arg38 (see Figure 1).^{8, 93} However, mutagenesis studies on Arg38 of yeast iso-1-Cytc⁹⁵ and the recent work on the G41S and Y48H variants suggest that HP7 may be the propionate with the pK_a > 9. Given the interconnectedness of the buried hydrogen-bond network, it has been suggested that loss of a proton occurs from the network rather than from a specific group.⁶² Regardless of the exact source of the proton, the ionization corresponding to pK_H appears to be more facile in THC4-linked Hu Cytc variants enhancing access to the alkaline state. This decrease in the pK_H of the group that triggers the alkaline transition also likely makes alternate conformers of Cytc that are nearby in energy to the native state more accessible above pH 7 so they can promote peroxidase activity.^86–87 The strong increase in peroxidase activity of the A51V variant relative to that of WT Hu Cytc at pH 8 is consistent with this contention.

Native-state H/D-exchange studies on horse Cytc,⁶⁸ show that the stability of Ω-loop C decreases from a stability of ~4 kcal/mol at pH 7 to a stability of 2.5 – 3 kcal/mol at pH 5. The decrease in the stability of Ω-loop C across this pH range has been linked to protonation of either His26 or a heme propionate,⁶⁸ presumably HP6. H/D exchange studies on the G41S and Y48H variants,^19–20 indicate that this loop is destabilized. The greater susceptibility of the A51V variant to acid unfolding (Fig. 7, Table 4) may also be linked to destabilization of Ω-loop C. The lower local stability of Ω-loop C may lead to functionally-significant population of peroxidase-competent conformers for the THC4-linked variants of Hu Cytc as pH drops from 7 to 5 and His26 or a heme propionate is protonated.

CONCLUSION

Studies on A51V Hu Cytc, like the other THC4-linked variants of Hu Cytc, G41S and Y48H, provide considerable insight into the mechanism by which Cytc accesses peroxidase-competent conformers important in the early stages of apoptosis. All have lower global and local stability than that of WT Hu Cytc. The A51V variant also is less stable to acid unfolding. The kinetics of the alkaline conformational transition indicate that enhanced access to the heme crevice needed for peroxidase activity results from a decrease in the pK_H of the trigger group for the alkaline conformational transition. The decrease in pK_H caused by these substitutions to Ω-loop C may be due changes in the environment of HP7 or more broadly to their effects on release of a proton from the buried H-bond network of Hu Cytc. By contrast, substitutions in Ω-loop D appear not to affect the pK_H of the triggering ionization and thus must destabilize Ω-loop D if enhanced peroxidase activity is to result. For A51V, the pK_H decreases for only one alkaline conformer, suggesting that the dynamics of the loop are bipartite and have different triggering ionizations. A similar bipartite effect on the stability of Ω-loop D is observed for Ω-loop D variants.⁶⁴ The enhancement of peroxidase activity below pH 7 appears to result from destabilization of Ω-loop C and may be linked to protonation of HP6 or His26. The latter effect may be more important for understanding the role of Ω-loop C in biologically relevant peroxidase activity given the pH of the intermembrane space.