Proline96 of the copper ligand loop of amicyanin regulates electron transfer from methylamine dehydrogenase by positioning other residues at the protein-protein interface

Abstract

Amicyanin is a type 1 copper protein that serves as an electron acceptor for methylamine dehydrogenase (MADH). The site of interaction with MADH is a “hydrophobic patch” of amino acid residues including those that comprise a “ligand loop” that provides three of the four copper ligands. Three prolines are present in this region. Pro94 of the ligand loop was previously shown to strongly influence the redox potential of amicyanin, but not affinity for MADH or mechanism of electron transfer (ET). In this study Pro96 of the ligand loop was mutated. P96A and P96G mutations did not affect the spectroscopic or redox properties of amicyanin but increased the K_d for complex formation with MADH and altered the kinetic mechanism for the interprotein ET reaction. Values of reorganization energy (λ) and electronic coupling (H_AB) for the ET reaction with MADH were both increased by the mutation, indicating that the true ET reaction observed with native amicyanin was now gated by or coupled to a reconfiguration of the proteins within the complex. The crystal structure of P96G amicyanin was very similar to that of native amicyanin but notably, in addition to the change in Pro96, the side-chains of residues Phe97 and Arg99 were oriented differently. These two residues were previously shown to make contacts with MADH that were important for stabilizing the amicyanin-MADH complex. The values of K_d, λ and H_AB for the reactions of the Pro96 mutants with MADH are remarkably similar to those obtained previously for P52G amicyanin. Mutation of this proline, also in the hydrophobic patch, caused reorientation of the side chain of Met51, another reside that interacted with MADH and caused a change in the kinetic mechanism of ET from MADH. These results show that proline residues near the copper site play key roles in positioning other amino acid residues at the amicyanin-MADH interface not only for specific binding to the redox protein partner, but also to optimize the orientation of proteins for interprotein ET.

Transition metals are used in proteins as structural elements and components of catalytic and redox-active centers (1). Several proteins contain copper which participates in a variety of biological functions ranging from activation of dioxygen to mediation of electron transfer (ET). Copper sites in proteins have been classified according to their spectroscopic and functional properties as type 1, type 2 or type 3 (2). A type1, or blue copper, site is so-named for an intense absorption band near 600 nm (ε ~ 5,000 M⁻¹ cm⁻¹) attributable to a cysteine S to Cu(II) charge transfer (3). Blue copper sites function as efficient ET mediators, and their oxidation-reduction midpoint potential (E_m) values span a wide range from +200 to +1,000 mV (4). Amicyanin (5) is a type 1 copper protein that is involved in metabolism of methylamine by several methylotrophic and autotrophic bacteria. In Paracoccus denitrificans, amicyanin mediates ET from methylamine dehydrogenase (MADH) (6) to cytochrome c_551i (7), and these electrons are ultimately donated to the membrane-bound respiratory chain. A crystal structure of the complex of MADH, amicyanin and cytochrome c_551i has been determined (8), and in the crystalline state the complex is catalytically active (9, 10) and transfers electrons from the tryptophan tryptophylquinone (TTQ) (11) cofactor of MADH to the cytochrome heme via the copper site of amicyanin. While MADH, amicyanin and cytochrome c_551i are isolated as individual soluble proteins, it has been demonstrated that in solution they must form a ternary complex to catalyze methylamine-dependent cytochrome c_551i reduction (12, 13). The ET reactions to (14–17) and from (18) the type I copper center of amicyanin within the protein complex have been studied in solution by stopped-flow spectroscopy. Analysis by ET theory (19) of the temperature dependencies of these ET reactions yielded values for the reorganization energy (λ), electronic coupling (H_AB) and distance between donor and acceptor that are associated with each of these ET reactions.

The structure of P. denitrificans amicyanin exhibits a β-sandwich topology with nine β-strands forming two β-sheets (20). The copper center possesses four coordinating ligands when oxidized: two N^δ of His52 and His95, S of Cys92, and S of Met98 at a longer distance forming a distorted tetrahedral geometry (Figure 1A). His95, Cys92, and Met98 are located in a “ligand loop” between two β-strands. The crystal structure of the protein complex reveals that both hydrophobic and electrostatic interactions involving several amicyanin residues are important in stabilizing the protein complex (20). The hydrophobic patch of amicyanin which interacts with MADH includes residues Met28, Met51, Pro52, Met71, Pro94, Pro96 and Phe97. Arg99 which is at the periphery of this patch is also important as it forms a salt bridge to MADH (Figure 1B). The ligand loops of several type 1 copper proteins (also called cupredoxins) have been structurally characterized (Table 1). The ligand loop is a common feature and believed to contribute to the specificity for associations with redox partner proteins (21, 22). Furthermore, residues in this loop contribute to an array of interactions, including hydrogen-bonding contacts which are thought to be critical for properties of the copper site. The length, sequence and structure of this loop varies in cupredoxins (21). Amicyanin contains the shortest ligand loop and is unusual in that the loop contains two proline residues, Pro94 and Pro96, which are also components of the hydrophobic patch. The presence of a proline between the Cys and His ligands in the ligand loop (Pro94 in P. denitrificans amicyanin) is a conserved feature seen in other cupredoxins. However, in the major classes of cupredoxins, a second proline between the His and Met ligands in the loop (Pro 96 in P. denitrificans amicyanin) is only seen in amicyanin. Furthermore, in P. denitrificans amicyanin Pro94 and Pro96 immediately flank the His95 ligand, a structural feature unique among cupredoxins.

Figure 1.

The structure of native amicyanin. A. The secondary structure of amicyanin is shown with the ligand loop highlighted in light blue and enlarged in the circle. B. A space-filling model of amicyanin with the residues that comprise the hydrophobic patch indicated in red and Arg99 which forms a salt-bridge with MADH colored green. Copper is shown as a blue sphere.

Table 1.

Comparison of Sequences of the C-Terminal Ligand-Containing Loops of the Major Classes of Cupredoxins

Protein	Ligand-containing C-terminal loopsa
Amicyaninb	C T H F M
Azurinc	C T F G H S A L M
Pseudoazurind	C T H Y A M G M
Plastocyanine	C S H Q G A G M
Stellacyaninf	C G V K H C D L G Q
Rusticyaning	C Q I G H A A T G M

^aLigands of copper are indicated in bold and Proline is marked in red;

^bParacoccus denitrificans (54);

^cPseudomonas aeruginosa (55);

^dAlcaligenes faecalis (56)

^ePopulus nigra (57);

^fRhus verniciflua (58);

^gThiobacillus ferrooxidans (59).

The conserved Pro94 was previously altered by site-directed mutagenesis (23, 24), and structural and mechanistic studies of P94F and P94A amicyanins revealed that the E_m value was made more positive due to alteration of H-bonding interactions of copper ligands (23). Mutation of another proline present in the hydrophobic patch, Pro52, resulted not only in loss of interactions of the Pro with MADH and but also repositioning of Met51 which altered the protein-protein interface. This decreased the binding affinity and altered the kinetic mechanism of the ET reaction with MADH (25, 26). To gain more insight into the roles of proline residues of the ligand loop and hydrophobic patch of cupredoxins in their interactions with redox partner proteins, and interprotein ET reactions, Pro96 was altered by site-directed mutagenesis and the consequence on the structure and function of amicyanin was characterized.

Experimental procedure

Protein Purification

Previously described procedures were used to purify MADH (27) and native amicyanin (5) from P. denitrificans. The protein concentrations were calculated using known extinction coefficients for oxidized amicyanin (ε₅₉₅ = 4610 cm⁻¹ M⁻¹) and MADH (ε₄₄₀ = 26200 cm⁻¹ M⁻¹). P96A and P96G amicyanin were expressed in Escherichia coli and purified from the periplasmic fraction as described previously for recombinant wild-type amicyanin (28). P96G amicyanin was isolated with only partial copper occupancy of the metal site, with some of the site containing zinc instead. The zinc was removed and the protein was reconstituted with copper as described previously for other amicyanin mutant proteins (29, 30). The extinction coefficients of P96A and P96G amicyanin were determined by anaerobic reductive titration of the fully oxidized protein with a solution of sodium dithionite which had been standardized by titration with native amicyanin.

Site-Directed Mutagenesis of the Amicyanin Gene

Site-directed mutagenesis was performed on double-stranded pMEG201 (28), which contains the mauC gene which encodes amicyanin, using two mutagenic primers with the QuickChange Site-Directed Mutagenesis Kit (Stratagene). The oligonucleotide sequences used to construct the site-directed mutants are as follows: P96A, 5'-TGCACCCCGCATGCCTTCATGCGC-3' and its complementary DNA; P96G, 5'-TGCACCCCGCATGGCTTCATGCGC-3' and its complementary DNA. The underlined bases are those that were changed to create the desired mutation. The entire 555-base mauC-containing fragment was sequenced to ensure that no second site mutations were present, and none were found.

Spectrochemical Redox Potential Determination

The E_m values of P96A and P96G amicyanins were determined by spectrochemical titration as described previously for native amicyanin (31). The ambient potential was measured directly with a redox electrode which was calibrated using quinhydrone (a 1:1mixture of hydroquinone and benzoquinone) as a standard with an E_m value of +286 mV at pH7.0 (32). The reaction mixture contained 130 µM amicyanin in 50 mM BisTris propane buffer at the indicated pH, at 25 °C. The mixture was titrated by addition of incremental amounts ascorbate, which was used as a reductant. In the oxidative direction, titration by addition of potassium ferricyanide was performed. The absorption spectrum of amicyanin was recorded at different potentials (E), and the concentrations of oxidized and reduced amicyanin were determined by comparison with the spectra of the completely oxidized and reduced forms. The E_m was determined from the fit of the data to eq 1.

$$E=E_m+(2.3\mathit{\text{RT}}/\mathit{\text{nF}})\text{ log }{[\text{amicyanin}]}_{\text{oxidized}}/{[\text{amicyanin}]}_{\text{reduced}}$$ (1)

For determination of the E_m value of amicyanin in complex with MADH, the reaction mixture contained 20 µM amicyanin and 60 µM MADH in 10 mM potassium phosphate buffer at pH 7.5, at 25 °C. Potassium ferricyaninde (400 µM), quinhydrone (200 µM), and phenanine ethosulfate (20 µM) were present as mediators. The absorption spectrum of the complex was recorded at different potentials and the concentrations of the oxidized and reduced forms were determined by comparison with the spectra of the completely oxidized complex and a mixture of oxidized MADH with fully reduced amicyanin as described previously (31). Data were then analyzed according to eq 1.

Electron Transfer Reactions between MADH and Pro96 Mutant Amicyanins

An On-Line Instruments (OLIS, Bogard, GA) RSM stopped-flow rapid scanning spectrophotometer was used for the kinetic measurements. Experiments were performed in a 0.01 M potassium phosphate buffer at pH 7.5. The experimental details and methods of data analysis have been previously described (33). To study the ET reaction from MADH to amicyanin, prior to mixing one syringe contained reduced (quinol) MADH, and the other contained varying concentrations of oxidized amicyanin. Pseudo-first-order conditions were maintained with excess amicyanin to MADH. ET reactions were fit to the simple kinetic model in eq 2 using eq 3.

$${\text{MADH}}_{\text{red}}+{\text{amicynin}}_{\text{ox}}\stackrel{K_d}{\rightleftharpoons }{\text{MADH}}_{\text{red}}-{\text{amicynin}}_{\text{ox}}\underset{k_4}{\overset{k_3}{\rightleftharpoons }}{\text{MADH}}_{\text{semi}}-{\text{amicynin}}_{\text{red}}$$ (2)

$$k_{\mathit{\text{obs}}}=k_{\mathit{3}}[\text{amicyanin}]/([\text{amicyanin}]+K_d)+k_{\mathit{4}}$$ (3)

Analysis of Electron Transfer Reactions by Electron Transfer Theory

The temperature dependence of k₃ was analyzed according to ET theory (19) (eq 4), where H_AB is the electronic coupling matrix element, λ is the reorganization energy, h is Plank’s constant, T is temperature, R is the gas constant, and k_o is the characteristic frequency of nuclei (10¹³ s⁻¹) which is the maximum ET rate when the donor and acceptor are in van der Waals contact and λ = −ΔG°. ΔG° is determined from the ΔE_m value for the reaction from eq 5, where F is the Faraday constant and n is the number of electrons transferred.

$$k_{\text{ET}}+[4{\pi }^2{H_{\mathit{\text{AB}}}}^2/h{(4\pi \lambda \mathit{\text{RT}})}^{0.5}]\text{exp}[-{(\mathrm{\Delta }{G}^o+\lambda )}^2/4\lambda \mathit{\text{RT}}]$$ (4)

$$\mathrm{\Delta }{G}^o=-\mathit{\text{nF }}\mathrm{\Delta }{E}_m$$ (5)

Steady-state kinetics

Steady-state kinetic experiments were performed as described previously (34). For the MADH-catalyzed reduction of amicyanin by methylamine the assay mixtures contained 18 nM MADH and varying concentrations of amicyanin in 0.01 M potassium phosphate buffer at pH 7.5 at 30 °C. Activity was monitored by the change in absorbance caused by the reduction of amicyanin at 595 nm and the data were fit to eq 6.

$$v/[\text{MADH}]=k_{\mathit{\text{cat}}}[\text{amicyanin}]/(K_m+[\text{amicyanin}])$$ (6)

Electron Paramagnetic Resonance (EPR) spectroscopy

EPR spectra were recorded using a Bruker (Karlsruhe, Germany) X-band spectrometer equipped with an ER 4119HS high-sensitivity cavity at 20 K using an Oxford Instruments low-temperature system. The native and mutant amicyanins were present at a concentration of 300 µM in 10 mM potassium phosphate, pH 7.4, containing 5% glycerol. Each EPR spectrum was recorded under non-saturating conditions.

Structure Determinations by X-ray Crystallography

P96G amicyanin was dialyzed against 5 mM sodium monobasic/potassium dibasic phosphate buffer, pH 6.6, prior to crystallization trials as was done previously with native and other amicyanin mutants (30, 35). Crystals were grown using the sitting drop method (1.5:1.5 µl protein/reservoir). The protein solution contained ~13 mg/ml protein, 5 mM sodium monobasic/potassium dibasic phosphate buffer, pH 6.6. The reservoir solution contained 1.8 M sodium/potassium phosphate, pH 7.5, including 40 mM ammonium sulfate. The crystal was briefly transferred to fomblin (purchased from Sigma-Aldrich) before freezing in liquid nitrogen and was maintained at 100 K during data collection.

Data sets were measured at a wavelength of 1.140 Å on an ADSC Q315 CCD detector at the 24ID-C beamline equipped with Microdiffractometer-MD2 at the NE-CAT, Advance Photon Source. The data were integrated, scaled and merged using HKL2000 (36). The structure of P96G amicyanin was solved by molecular replacement using the program PHASER (37) of PHENIX (38). A 1.3 Å resolution crystal structure of the native amicyanin (PDB code 1AAC) with Pro96 mutated to Gly was used as a search model. The resultant model was subjected to refinement using PHENIX (38) and a difference Fourier map (Fo-Fc) was calculated. The map confirmed the presence of Gly96. The model was subjected to several cycles of alternative positional and thermal parameters, including restrained TLS (Translation/Libration/Screw) refinement as implemented in PHENIX.REFINE (38, 39). The model was corrected and rebuilt wherever necessary using COOT (40). For cross validation (41), ~6% (1871 reflections) of all reflections were set aside from refinement for calculation of R_free. When the R and R_free were 31.2 and 33.7, respectively, water molecules were added to the model. A simulated annealing omit map was calculated around the copper region and Gly96 to remove any model bias. At no point in the refinement were restraints applied to the copper-ligand distances or bond lengths. The simulated annealing refinement was applied once to the model. The final stereochemistry of the model was checked against the Ramachandran map in PROCHECK (42). The refined model of P96G amicyanin has four molecules in the asymmetric unit. A summary of the data analysis, refinement results, and stereochemistry is presented in Table 2.

Table 2.

Data Collection, Refinement and Model Statistics for P96G Amicyanin Structure^a

	P96G amicyanin
Data Collection
Wavelength (Å)	1.140
Space group	P 3221
Unit Cell Dimension
a (Å)	97.3
b (Å)	97.3
c (Å)	109.9
resolution limit(Å)	50–2.2
beamline	NE-CAT 24ID-C
I/Sigma(I)	23.2(3.1)
Rmerge (%)	6.9(55.7)
Completeness (%)	99.8(100.0)
Redundancy	7.2
Refinement
resolution range (Å)	50–2.2
R-factor (%)	23.9
Rfree (%)	29.1
R-factor (Work + Test sets) (%)	24.3
No. of Reflections	28971
Model
No. of amino acids	420
No. of water molecules	180
No. of Copper	4
No. of potassium	1
No. of Sodium	2
No. of phosphate	2
Average B-factor (Ǻ2) for protein atoms	49.3
Residues in generously allowed regions	2
Residues in disallowed regions	0
No. of atoms with zero occupancy	71
Stereo chemical ideality
bonds (Å)	0.008
angles (°)	1.128
dihedral angles (°)	13.948

^aValues in parentheses are for the outer shell

Results

Spectroscopic Properties

The visible absorption spectra of P96A and P96G amicyanin are essentially the same as native amicyanin with an absorption maximum centered at 595 nm (Figure S1 and Table 3). The extinction coefficients of P96A and P96G amicyanin were determined to be 4.6 mM⁻¹cm⁻¹ and 4.9 mM⁻¹cm⁻¹, respectively, as compared to 4.6 mM⁻¹cm⁻¹ for native amicyanin. The EPR spectra of oxidized P96A and P96G amicyanins were also nearly identical to that of native amicyanin (Figure S2 and Table 3). For P96A amicyanin the spectra were of the as-isolated protein which has full occupancy of copper. For P96G amicyanin the protein was isolated with zinc present in a large fraction of the protein and the spectra shown here are of the protein after removal of zinc and reconstitution with copper.

Table 3.

Spectroscopic and Redox Properties of Native, P96A and P96G Amicyanins

	Native amicyanin	P96A amicyanin	P96G amicyanin
Absorption λmax (nm)	595	595	595
ε595 (cm−1 mM−1)	4.6	4.6	4.9
EPR g⊥	2.06	2.05	2.05
EPR g‖	2.24	2.24	2.24
EPR A‖ (G)	53	52	51
Em at pH 7 (free) (mV)	294 ± 7a	285 ± 1	311 ± 1
Em at pH 7 (in complex) (mV)	224 ± 10a	241 ± 2	229 ± 3

^aTaken from ref (31).

Redox Properties

The redox properties of P96A and P96G amicyanin were determined by spectrochemical titration of each protein, free and in complex with MADH (Table 3). The E_m values of free P96A and P96G amicyanin at pH 7.0 are 9 mV less and 17 mV more positive than native amicyanin, respectively. The E_m value of native amicyanin in complex with MADH is known to decrease on complex formation with MADH because a redox state-dependent conformational change that is linked to the protonation of His95 is sterically constrained in the complex relative to free amicyanin (31). This E_m value of amicyanin in complex with MADH is essentially that of free amicyanin in the pH-independent region (> 9.0) and is used for calculation of ΔG° when analyzing ET reactions which occur within the protein complex. The E_m value of P96G amicyanin in complex with MADH was within error of the value in the native complex, and the E_m value of P96A amicyanin in complex with MADH was slightly more positive than in the native complex (Table 3). Thus, these mutations of Pro96 have a minimal effect on the redox properties of amicyanin.

Steady State Reactions between MADH and Pro96 Amicyanin Mutants

The steady-state kinetics of methylamine-dependent amicyanin reduction by MADH was examined (Figure 2 and Table 4). P96A and P96G amicyanins exhibited k_cat values of 26 s⁻¹ and 8 s⁻¹, respectively, compared to 61 s⁻¹ for native amicyanin. The K_m value for each mutant was also significantly larger than for native amicyanin. Overall, the P96A and P96G mutations decreased the values of k_cat/K_m by 16-fold and 47-fold, respectively.

Figure 2.

Steady-state kinetic analysis of the methylamine-dependent reduction of P96A (A) and P96G (B) amicyanin by MADH. Reaction conditions are described under Experimental Methods. Lines are fits of the data to eq 6.

Table 4.

Steady State Kinetic Parameters

	Native amicyanina	P96A amicyanin	P96G amicyanin
Km (µM)	1.3 ± 0.3	8.9 ± 0.4	6.4 ± 0.3
kcat (s−1)	61 ± 6.0	26.4 ± 0.4	7.9 ± 0.1
kcat/Km (M−1s−1)	4.7 × 107	0.3 × 107	0.1 × 107

^aTaken from ref (51)

Electron Transfer Reactions between MADH and Pro96 Amicyanin Mutants

The ET reactions from quinol MADH to P96A and P96G amicyanins were also studied under single-turnover conditions. Values of K_d and k₃ (see eq 2) were obtained from analysis by eq 3 of the dependence on amicyanin concentration of the observed rate of reaction from quinol MADH (Figure 3 and Table 5). The K_d values of P96A and P96G amicyanin were increased approximately 10-fold and 8-fold, respectively. While the ΔG° values for the ET reactions from quinol MADH to P96A and P96G amicyanin are slightly more favorable than for native amicyanin, the k₃ values for P96A and P96G amicyanins were decreased by 2-fold and 5-fold, respectively. As noted in Table 5, these k₃ and K_d values of P96A and P96G amicyanin are remarkably similar to those previously reported for another mutant, P52G amicyanin (43) (discussed later).

Figure 3.

Concentration dependence of the observed rate of the ET reaction from MADH to P96A (A) and P96G (B) amicyanin. Reaction conditions are described under Experimental Methods. Lines are fits of the data to eq 3.

Table 5.

Electron Transfer Parameters for the Reactions of Quinol MADH with Native and Mutant Amicyanins

Parameters	Native amicyanina	P96A amicyanin	P96G amicyanin	P52G amicyaninb	P94F amicyaninc
Kd (µM)	4.5	47	36	38	<5
k3 at 30°C (s−1)	10	5	2	3	60
ΔG° (kJ mol−1)	–3.18	–4.83	–3.47	–4.82	–21.7
λ (eV)	2.3 ± 0.1	2.7 ± 0.1	2.8 ± 0.1	2.8 ± 0.1	2.3 ± 0.1
HAB (cm−1)	12 ± 7	42 ± 15	51 ± 14	78 ± 30	4.6 ± 1.3

^aTaken from (14).

^bTaken from (43).

^cTaken from (23, 46).

To determine the values of H_AB and λ that are associated with these ET reactions, the k₃ values for the ET reaction from quinol MADH to P96A and P96G amicyanins were determined over a range of temperatures and the data were analyzed using eq 5 (Figure 4). Analysis of these data revealed that the values for both λ and H_AB are significantly increased for the reaction with each mutant amicyanin (Table 5). This trend is characteristic of a change in the kinetic mechanism of the redox reaction such that it is no longer a true ET reaction (44, 45). The values of H_AB, and λ which were obtained were also similar to the parameters that were previously reported for the ET reactions from quinol MADH to P52G amicyanin (Table 5) (25). In contrast, the previously reported P94A and P94F amicyanin mutations primarily affected the E_m value, and consequently ΔG^o, but did not significantly increase H_AB or λ (Table 5) (23, 46).

Figure 4.

Temperature dependence of the observed limiting first order rate constant for the reactions of reduced MADH with oxidized to P96A (circles) and P96G (triangles) amicyanin. Reaction conditions are described under Experimental Methods. Lines are fits of the data to eq 4.

Crystal Structure of P96G amicyanin

The structure of oxidized P96G amicyanin was determined at 2.2 Å resolution. P96G amicyanin crystallized in the hexagonal space group P3₂21, with unit cell parameters a=b=97.3, c=109.9. This space group is different from those of previously crystallized native and mutant amicyanins (30, 47). The refined model of P96G amicyanin has four molecules in the asymmetric unit which together comprise 420 amino acids with 180 water molecules, four coppers, two phosphate, two Na and one K ions. There were 71 atoms (7, 6, 11 and 47 in molecules A, B, C and D, respectively) which did not have electron density and have been assigned zero occupancy. The average temperature factors (Ų) for the protein molecules A–D are 64, 46, 60 and 122, respectively while it is 60 for all the water molecules. As Molecule B exhibited the lowest temperature factor and highest quality of density of the four molecules, subsequent analysis was performed with Molecule B.

In general, the structure of P96G amicyanin varies little from that of the native amicyanin. The rms deviation between native (PDB entry 2OV0) and Molecule B of P96G amicyanin is 0.62 Å with 103 matched C^α atoms based on the secondary structure matching (48) calculated with CCP4MG (49) (Figure 5). The difference between native and P96G is most pronounced in the region 15–21. However, it was observed earlier that N-terminal residues 1–21 comprise a very flexible loop segment that adopts variable conformations (23). By omitting this segment, the rms deviation drops to 0.29 Å. The mutation caused no significant change in the type 1 copper site and the copper coordination distances in the P96G amicyanin are very similar to those in native amicyanin (Table 6).

Figure 5.

Stereoview diagram of the superposition of P96G amicyanin (red) with native amicyanin (blue). The C^α trace of protein chains are displayed with residues numbered at 10-residue interval and copper shown as a blue sphere.

Table 6.

Copper Coordination Distances in the Type 1 Sites of P96G and Native Amicyanin

Ligands	P96G amicyanina	Native amicyaninb
Cu - SG/Cys92 (Å)	2.32	2.17
Cu - ND1/His95 (Å)	2.12	2.05
Cu - ND1/His53 (Å)	1.96	1.99
Cu – SD/Met98 (Å)	2.93	3.07

^aData for Molecule B,

^bData from PDB entry 2OV0

While the overall structure of amicyanin was unchanged as a consequence of the P96G mutation, significant changes were observed in the orientation of residues which have been shown to stabilize the amicyanin-MADH complex. The side-chain of Phe97 deviates by ~1Å. Comparison of the structure of P96G amicyanin with that of native amicyanin in the ternary protein complex reveals that this change in position leads to weakening of the van der Waals’ interaction between Phe97 and αArg184 of MADH (28) (Figure 6). The importance of Phe97 in the interaction with MADH was demonstrated previously as a F97E mutation increased the K_d for complex formation (28) and decreased the rate constant for ET from MADH to amicyanin (50).The position of the side-chain of Arg99 has also changed as a consequence of the P96G mutation such that it is no longer in the proper orientation to form the salt bridge with αAsp167 of MADH that is observed in the structure of the native protein complex (Figure 6). The importance of this ionic interaction towards stabilizing the amicyanin-MADH complex was previously demonstrated by site directed mutagenesis of Arg99 (28) and αAsp167 (51). The P96G mutation also eliminates the hydrophobic interactions of Pro96 with MADH.

Figure 6.

Stereview of superposition of P96G amicyanin with native amicyanin within MADH-amicyanin-cytochrome c-551i complex (PDB code 2MTA). The α and β subunits of MADH are colored blue and shown as ball and stick. Selected residues of P96G amicyanin (red) and native amicyanin (black) are shown as ball and stick with the copper shown as a sphere.

Discussion

Despite the lack of significant effects of the P96A and P96G mutations on the structure of the type 1 copper site, and spectroscopic and redox properties of amicyanin, these mutations had a significant effect on the rate and mechanism of the ET reaction from MADH, in addition to increasing the K_d for complex formation. The crystal structure of the native MADH-amicyanin complex reveals that Pro96 is present at the protein-protein interface (8, 52). Consistent with that observation, replacement Pro96 with Ala or Gly increased the K_d values for complex formation 10-fold and 8-fold, respectively. The mutations also caused decreases in the apparent ET rate constant (k₃), which is independent of K_d. Analysis of the temperature dependence of k₃ revealed that the mutations caused significant increases in the experimentally-determined values of values H_AB and λ. This trend is characteristic of a change in kinetic mechanism from a true ET reaction to one which is either gated or coupled (45) (Scheme 1). Gated electron transfer occurs when a relatively slow adiabatic (non-electron transfer) reaction precedes and is required to activate the system for a faster nonadiabatic (true electron transfer) reaction. For a gated ET reaction the observed rate constant for the reaction will actually be the rate constant for the preceding slower non-ET reaction step. Coupled electron transfer is a process in which a relatively rapid but thermodynamically unfavorable non-electron transfer reaction precedes, and is required to activate the system for a relatively slow electron transfer reaction. Since the activated and unreactive states exist in an equilibrium that favors the unreactive state, the observed rate constant (k_obs) for the redox reaction is an apparent value equal to the product of the true electron transfer rate constant (k_ET) and the equilibrium constant for the preceding non-ET reaction step.

Scheme 1.

Kinetic mechanisms of electron transfer reactions

The values for K_d, k₃, H_AB, and λ which were determined for the reactions of P96A and P96G amicyanin were very similar to those that were previously reported for the ET reactions from quinol MADH to P52G amicyanin (43) (Table 5). A consequence of the P52G amicyanin mutation was also that it altered the position of another residue, Met51. Subsequent study of Met51 amicyanin mutants revealed that the loss of atoms of Pro52 in P52G amicyanin was only responsible for the change in K_d, while the change in position of Met51 in P52G amicyanin was entirely responsible for the change in ET parameters (26). Since interactions between the Met51 side-chain and MADH were lost as a consequence of the M51A mutation, it was concluded that the mutation slowed the rate of normally rapid conformational rearrangement that precedes ET resulting in conversion of the true ET reaction to a gated ET reaction. These same ET reactions from MADH to native and M51A amicyanin were recently studied using molecular dynamics simulations to estimate the ET coupling strength of ensembles of configurations of the native and mutant protein complex (53). It was concluded that hydrophobic residues surrounding the acceptor site on amicyanin comprised a “molecular breakwater” that supports the function of nearby solvent-organizing residues by limiting the exchange of water molecules between the sterically constrained ET region and bulk solvent. It was shown that mutation of Met51disrupted this breakwater resulting in a decreased availability of the optimum configuration for ET, consistent with the previous experimental results of the effects of this mutation on ET (26).

While it may be coincidental, the very similar effects of the Pro96 amicyanin mutations on λ and H_AB to those of the P52G and M51A amicyanins, suggest that the Pro96 mutations have perturbed the kinetic mechanism for the ET reaction from MADH in a similar manner to that of the P52G mutation. This suggestion is supported by the similar nature of the structural changes that are caused by the respective mutations. As was shown for Pro52, the presence of Pro96 is critical for positioning other residues at the MADH-amicyanin interface. The results indicate that subtle changes in the protein-protein interactions at the MADH-amicyanin interface involving any of multiple residues can cause either a decrease in the relative rate of a configurational rearrangement that is required subsequent to complex formation to poise the system for ET, or a decrease in the availability of an optimum configuration for ET from an ensemble of configurations, or both. In either case this alters the observed rate constant for the ET reaction as a consequence of altering the kinetic mechanism of the overall reaction (see Scheme 1). These results highlight the fact that specific individual residues at the surface of redox proteins not only dictate specificity for their redox protein partners, but are critical to optimize the configuration of the redox centers and intervening media within the protein complex for the ET event.

The results of these studies also highlight and further define the multiple roles of amino acid residues within the ligand loop of type 1 copper proteins. Previous structural and mechanistic studies of mutations of Pro94, a proline whose presence is conserved in the ligand loops of cupredoxins (Table 1), revealed an important role for this residue in controlling the E_m value of amicyanin. It was shown that mutations of Pro94 to other amino acids resulted in formation of an electron-withdrawing hydrogen bond to the copper-coordinating thiolate sulfur of Cys92 which was introduced by movement of the amide N much closer to the thiolate sulfur than in native amicyanin (23). Thus, the conserved proline in this position in the ligand loop is likely required to maintain an appropriate hydrogen bonding environment for the strong Cys ligand which influences the E_m value. The role of Pro96, a proline whose presence is not conserved in the ligand loops of cupredoxins, is completely different and specifically related to the physiological role of amicyanin. Proline at this position has no effect on the electronic and redox properties of amicyanin. Rather it plays key roles in recognition of its redox partner, as evidenced in alteration of K_d values, and in optimization in the configuration of the redox centers within the complex for ET, as evidenced by alteration of ET parameters diagnostic of a change in the kinetic mechanism of the overall redox reaction. A similar, amicyanin-specific role for a proline was previously identified for Pro52 (25). In both the Pro96 and Pro52 mutant amicyanins the effects on the ET reaction mechanism were due to alteration of positions of other residues that interacted with MADH. These results demonstrate how proline residues can significantly affect protein-protein interactions as a consequence of local changes in the structure of the protein backbone which effect the orientations of side chains of nearby amino acid residues. The demonstration of this “indirect” role of proline residues in dictating the specificity of protein-protein interactions is likely not limited to interactions between redox proteins but may be a general feature of specificity of interactions between proteins.