Exploring the mechanisms of the reductase activity of neuroglobin by site-directed mutagenesis of the heme distal pocket

Abstract

Neuroglobin (Ngb) is a six-coordinate globin that can catalyze the reduction of nitrite to nitric oxide. Although this reaction is common to heme proteins, the molecular interactions in the heme pocket that regulate this reaction are largely unknown. We have shown that the H64L Ngb mutation increases the rate of nitrite reduction by 2000-fold compared to the wild-type Ngb (Tiso, M. et al. (2011) J. Biol. Chem. 286, 18277–18289). Here we explore the effect of distal heme pocket mutations on nitrite reduction. For this purpose, we have generated mutations of the Ngb residues Phe28(B10), His64(E7) and Val68(E11). Our results indicate a dichotomy in the reactivity of deoxy five- and six-coordinate globins towards nitrite. In hemoglobin and myoglobin there is a correlation between faster rates and more negative potentials. However, in Ngb, reaction rates are apparently related to the distal pocket volume, and redox potential shows a poor relationship with the rate constants. This suggests a relationship between the nitrite reduction rate and heme accessibility in Ngb, particularly marked for His64(E7) mutants. In five-coordinate globins His(E7) facilitates nitrite reduction, likely through proton donation. Conversely, in Ngb the reduction mechanism does not rely on proton delivery from the histidine side chain, as His64 mutants show the fastest reduction rates. In fact, the rate observed for the H64A Ngb (1120 M⁻¹s⁻¹) is to our knowledge the fastest reported for a heme nitrite reductase. These differences may be related to a differential stabilization of the iron-nitrite complexes in five- and six-coordinate globins.

Six-coordinate globins are a group of heme proteins with high structural similarity to five-coordinate globins like hemoglobin (Hb) and myoglobin (Mb). However, as a notable difference with five-coordinate globins the distal histidine residue is bound to the heme iron in the ferrous (Fe^II) and usually in ferric (Fe^III) states, yielding a six-coordinated iron. In these proteins, distal histidine dissociation is required to allow binding of ligands to the heme iron. As pointed out, the distal histidine (E7 in general globin nomenclature) in five-coordinate globins is not in direct contact with the iron atom, but plays a critical role in ligand stabilization. A vast literature has explored the importance of this and other residues in the heme distal pocket of Mb with regards to ligand binding, heme autoxidation and other properties ^1–3. Whereas we can expect that six-coordinate globins recapitulate some of the observed behaviors, there is limited information about the role of these residues in six coordinate globins, and notable differences between both globin families may exist. As an example, we have observed very different behaviors in the nitrite reduction rates of His(E7) mutations ⁴. A better understanding of the structure-function relationships in six-coordinate globins is important for the elucidation of the often unknown function of these proteins.

The reduction of nitrite to nitric oxide (NO) is a reaction of important physiological consequences that can overcome the decrease in activity of NO synthases under hypoxic conditions ⁵. Nitrite reduction by hemoglobin has been proposed to regulate blood pressure, hypoxic vasodilation, platelet activation and the cellular resilience to hypoxia ^6–9. Among the proteins catalyzing this reaction, special emphasis has been put on the role of heme proteins ^{10, 11}, with most studies involving the reaction of Hb ^12–14. We have previously studied the nitrite reductase activity of other six-coordinated globins ^{4, 15}. Our results indicate that the reactivity of the proteins with nitrite spans in at least three orders of magnitude, and –at least on the case of neuroglobin- can increase up to 2500 fold by the removal of the histidine 64 (E7) side chain. The rates of nitrite reduction by wild-type six-coordinate globins appear to be higher than these of five coordinate globins, and vary in a wide range ^{4, 15–17}. Different hypothesis have been proposed to explain this faster rates in six-coordinated globins and distal histidine mutants. Factors like heme accessibility or redox potential have been related to the nitrite reductase rates ^{4, 14}. Studies on Ngb and other globins have shown the ability of heme distal pocket residues to modulate these and other heme properties ^{4, 18–23}. Here we present a detailed study of distal pocket mutations in the positions Leu29 (B10), His64 (E7) and Val 68 (E11) (Figure 1) in order to elucidate the more relevant factors that modulate nitrite reduction. Our study aims to orientate further work in six-coordinate globins to engineer proteins with tailored nitrite reductase activities.

Figure 1. Location of selected heme pocket residues.

The relative location of the neuroglobin residues studied in this work is shown. Top panel, side view; Bottom panel, top view. Heme moieties and side chains are shown as sticks (Ngb Phe28, blue; Ngb His64, red; Ngb Val68, green; Ngb His96 and Ngb heme, yellow; Mb Leu29, light blue; Mb His64, light red; Mb Val68, light green; Mb His93 and Ngb heme, pale yellow). The proximal histidines (Mb His93/Ngb His96) are omitted in the top view for clarity. The PDB files used are 1OJ6 (Ngb) and 2W6W (Mb).

MATERIALS AND METHODS

Reagents and protein preparation

All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. UV-visible spectra and kinetic data were recorded on an HP8453 UV-Vis spectrophotometer (Agilent Technologies, Palo Alto, CA) or a Cary 50 spectrophotometer (Agilent Technologies, Palo Alto, CA).

Cloning, expression and purification of recombinant Ngb

Molecular biology was performed using standard techniques. The previously used pET28-Ngb plasmid encoding a His tagged protein and a thrombin cleaving site ⁴ was modified so the Ngb coding sequence (originally inserted between the EcoRI and HindIII sites) was inserted between the NcoI and HindIII sites and the protein was expressed without a His tag. The wild-type Ngb plasmid generated was used as a template for the site directed mutagenesis reactions to produce the F28W, F28L, F28V, F28H, H64W, H64Q, H64A, H64L, V68A, V68F and V68I mutants using the Quikchange II site-directed mutagenesis kit (Stratagene, Palo Alto, CA) with the adequate primers. The sequences were confirmed by DNA sequencing at the University of Pittsburgh Genomics and Proteomics Core. The plasmids were transformed into SoluBL21 E. coli cells (Genlantis).

Purification of Ngb proteins was carried out based on reported methods with a number of modifications ^{4, 24}. Initial cultures were grown overnight at 37°C in LB broth containing 30 μg/ml kanamycin. The cultures were transferred to 4 liter flasks containing 1 liter of TB (50 ml of initial culture added per liter of TB) and grown at 37°C until OD 600nm = 0.8. Then 0.4 mM δ-amino-levulinic acid was added to the media and protein expression was induced with 1 mM IPTG. Protein expression was continued for 20–24 hours at 37°C. Cells were harvested and either lysed or kept at -80C until processed. Cells were lysed in 50 mM MOPS buffer, pH 7.0 containing 1 mM EDTA, 1 mg/ml lysozyme, 1 mM PMSF and 0.5 DTT. The cell lysis was accomplished by sonication (8–12 pulses of 30s at 35% amplitude) using a Misonix S-4000 sonicator (Qsonica, Newtown, CT). The crude lysate was clarified by centrifugation at 20,000 × g for 60 minutes. In order to remove nucleic acids from the sample, polyethyleneimine was added to the clarified supernatant to a final concentration of 0.1% (v/v). The precipitated nucleic acids were removed by centrifugation at 8,000 × g for 10 minutes. The supernatant was then loaded into a DE-32 column (Whatman) equilibrated with 50 mM MOPS, pH 7.0, 10 mM NaCl. The protein was eluted with a gradient from 10 to 100 mM NaCl in 50 mM MOPS, pH 7.0. After the DE-32 anion exchange column, the pooled protein fractions were passed through a Amicon Ultra centrifugal filter (Millipore) with a 50 kDa cutoff to remove high molecular weight contaminants. The flowthrough was concentrated using a 10 kDa cutoff Amicon Ultra centrifugal filter (Millipore) and buffer exchanged to 100 mM phosphate buffer, pH 7.4. This procedure generally yielded protein samples with >85% purity as assessed from SDS-PAGE electrophoresis. Alternatively, instead of using the 50 kDa filters, some samples were further purified by gel chromatography. In these cases, after the DE-32 column samples were concentrated using a 10 kDa cutoff Amicon Ultra centrifugal filter (Millipore). The concentrated sample (2–3 ml) was loaded into a Sephacryl S200HR column (GE Healthcare) equilibrated with 100 mM phosphate buffer, pH 7.4.

Protein chromatography steps were carried out using a ÅKTA Purifier 10 FPLC system (GE Healthcare) with UNICORN software. Protein purity was assessed by SDS-PAGE and UV-visible spectroscopy.

Nitrite reduction experiments

The reactions were carried out anaerobically in 3.5 ml optical glass cuvettes (Starna Cells, Atascadero, CA) closed by a screw cap with a silicone septum. Reactions were followed at 37 °C in Sodium Phosphate 100 mM, pH 7.4. The experiments were conducted in the presence of 2.5 mM sodium dithionite. Under these conditions the reduction of met-Ngb proceeds at rates around 20 s⁻¹. The observed rate of Ngb-NO formation in the reactions is below 0.2 s⁻¹. Reactions were initiated by addition of sodium nitrite from an anaerobic stock solution (1–100 mM) to yield the desired final concentration of nitrite (50 μM - 10mM).

Autoxidation rates

Preparation of the oxy-Ngb (Fe^II-O₂) species from the met-Ngb (Fe^III) samples was as follows: samples of wild-type or mutant neuroglobins (10–50 μM initial concentration) were reduced to the deoxy–Ngb form with excess dithionite at room temperature and then passed through a Sephadex G25-column (PD10, GE Healthcare) equilibrated with sodium phosphate buffer (100 mM, pH 7.4) to remove dithionite. Due to the high oxygen affinity of the protein, this step both removed the excess dithionite and produced, by reaction with oxygen in the buffer, quantitative formation of the oxy-Ngb (Fe^II-O₂). The protein was collected after the column and mixed with buffer at 37 °C in a 1:2 protein:buffer ratio to ensure an initial temperature around 37 °C. Due to the fast oxidation of some neuroglobins, significant oxidation of the oxy-Ngb during the column filtration step occurred in some cases. For the mutants with faster autoxidation rates, the deoxy-Ngb (Fe^II) species was prepared by dithionite reduction as described above, but the process was carried out in an anaerobic glove-box. The sample was then transferred to a sealed cuvette and the reaction was started by addition of aerobic buffer. Reaction rates were followed at 37 °C in a Cary 50 spectrophotometer with a thermostated cell holder. Spectral changes were monitored between 450 and 700 nm for slower reactions or between 500 and 600 nm for fast reactions. Scans were taken every 12s or every 6s at a scan rate of 2400 nm/min. The wavelengths showing the maximum absorbance change between oxy-Ngb and met-Ngb species were used to determine the autoxidation rates. Spectral changes were fit to a single exponential equation using the Origin 8.0 software (OriginLab Corporation, Northampton, MA).

Redox potentiometry

Redox titrations were performed inside a glove box (Coy Laboratory Products, Grass Lake, MI) under nitrogen atmosphere and 1–4% hydrogen to remove residual oxygen with a Pd catalyst. Neuroglobin samples (final concentration approximately 10 μM) were oxidized with potassium ferricyanide and then run through a Sephadex G25-column (PD10, GE Healthcare) equilibrated with anaerobic buffer to remove excess ferricyanide. Spectrophotometric measurements were carried out at 25 °C in 100 mM Sodium Phosphate buffer, pH 7.0. Phenazine methosulfate (E_m +80 mV), 2-methyl-1,4-naphtoquinone (E_m +10 mV), Indigo tetrasulfonate (E_m −46 mV), 2-hydroxy-1,4-naphtoquinone (E_m −137 mV) and Anthraquinone-2,6-sulphonate (E_m −184 mV) were used as redox mediators at concentrations between 1 and 5 μM. Potentials were determined with a MI-800/410 redox electrode (Microelectrodes Inc., Bedford, NH) coupled to an Accumet AB15 pH/mV meter. A correction factor of +199 mV at 25 °C was used for the electrode readings. The protein was titrated with sodium dithionite and the spectra monitored after each dithionite addition. The one-electron midpoint potentials were determined from the difference spectra. Fraction oxidized for each spectrum was calculated from the maximum difference between the oxidized and reduced spectra (around 430 nm and 390 nm). Using these data and the corresponding measured potentials (vs SHE) the midpoint potential of the half-reaction can be determined using the Nernst equation (equation 1):

$$E=E_{\mathrm{m}}+\frac{RT}{nF}2.303log\frac{[\text{Oxidized}]}{[\text{Reduced}]}$$ (1)

where E is the measured equilibrium potential at each titration point, R is the gas constant (8.314 J/mol K), T is the experimental temperature in Kelvin, n is the number of electrons in the half-reaction, F is the Faraday constant (96485 C/mol), and [Oxidized]/[Reduced] is the ratio of oxidized to reduced species.

Statistical Analysis

Data were analyzed using Origin 8.0 (OriginLab Corporation, Northampton, MA) and values are expressed as mean ± standard deviation of the mean.

RESULTS

Protein expression and purification

The wild-type and mutant proteins were overexpressed in E. coli as described in the methods section. We did not observe noticeable variations in the expression levels of the mutants as compared to the wild-type protein. The protein is generally purified in the oxidized form, however the His64 mutants (particularly H64Q and H64A) were purified mostly in the nitrosyl (Ngb Fe^II-NO) form as reported for other His64 mutants ²⁵ and consistent with intracellular scavenging of NO by these high affinity mutants.

Spectral properties

The mutations of the neuroglobin residues Phe28 (B10) and Val68 (E11) studied did not produce significant changes in the spectral properties of the deoxy-Ngb (Fe^II) and met-Ngb (Fe^III) species. As expected, mutation of the distal histidine residue His64 (E7) changes heme iron coordination form the wild-type six-coordinate form to a five-coordinate environment. This change is apparent in the spectra of the deoxy-Ngb (Fe^II) species, where the spectra of the six-coordinate heme with two peaks with maxima around 520nm and 550nm are shifted to a five-coordinated species with a single peak around 555nm, as observed for H64L and H64Q ^{4, 26, 27}. Remarkably, the spectrum of the deoxy-NgbH64A shows two peaks similar to those of the wild-type protein, and in the case of H64W a shoulder around 520 nm is observed. The origin of this “residual” 6-coordination is unknown but may be due to the stabilization of a solvent molecule in the heme pocket. A similar phenomenon has been described in some cases for Ngb H64L ^{21, 28}.

Autoxidation rates of mutant neuroglobins

We studied the autoxidation of human wild-type and mutant neuroglobins at 37 °C in 100 mM Sodium phosphate, 100 mM, pH 7.4. As previously shown, the oxygenated form of wild-type Ngb (oxy-Ngb, Fe^II-O₂) is unstable and decays, in a single exponential fashion, to form the oxidized, ferric form (Fe^III, met-Ngb) ²⁶ (Figure 2A). For wild-type neuroglobin, the oxy-Ngb species decays at a rate of autoxidation of 0.23 min⁻¹ at 37 °C (Table 1), not far from the value of 0.17 min⁻¹ obtained at pH 7.5 and 25 °C by Fago et al. ²⁹.

Figure 2. Autoxidation of Ngb wild type and His64 mutants.

The plots show selected spectra during the time course of autoxidation for each mutant. Arrows indicate the direction of the absorbance change. Insets show the fitting of the decay to a single exponential equation. A; Wild-type; B, H64A; C, H64Q, D, H64W.

Table 1.

Autoxidation rates of wild-type and mutant neuroglobins.

Ngb mutation	kautox (min−1)
Wild-type	0.23 ± 0.03
F28W	3.22 ± 0.42
F28L	1.76 ± 0.13
F28V	3.06 ± 0.22
F28H	11.3 ± 0.7
H64W	0.076 ± 0.006
H64Q	0.010 ± 0.002
H64A	0.066 ± 0.005
V68A	2.38 ± 0.21
V68F	0.039 ± 0.001
V68I	0.122 ± 0.005

All the His64 (E7) substitutions resulted in a decrease of autoxidation rates (Figure 2 B-D, Table 1). H64W and H64A induced a 3-fold decrease in autoxidation rates, whereas H64Q was less susceptible to autoxidation, with a more than 20-fold decrease in autoxidation rate. This result is in agreement with previous observations of Ngb mutants mentioning that H64Q and H64L/K67L were “remarkably stable in the oxygenated form” ²⁹.

Mutation of the Phe28 residue (B10) significantly decreases the stability of the oxy complex. As observed in Figure 3, the Phe28 mutants hardly form the Fe^II-O₂ complex, with peaks around 545 and 575 nm, and instead a mixture of deoxy-Ngb (with a peak around 560 nm) and oxy species is formed. The two species decay to the ferric form at apparently similar rates. Studies on myoglobin have shown that two mechanisms can be involved in heme autoxidation, an inner sphere, unimolecular autoxidation mechanism where the FeO₂ complex produces Fe^III and superoxide and an outer sphere, bimolecular autoxidation mechanism where the deoxy heme is oxidized by oxygen without formation of a Fe^II-O₂ complex. ¹⁸. Our results suggest that the autoxidation of the Phe28 mutants involves both mechanisms, with a significant contribution of the outer sphere electron transfer mechanism, and no formation of the Fe^II-O₂ complex. Partial formation of the oxy complex can be observed for F28V, F28W and F28L (Figures 3A, C & D). In the case of F28H (Figure 3B) we do not observe significant build-up of oxy species and the deoxy decays to met-Ngb in what appears to be a purely outer sphere electron transfer reaction . In all cases the rates of autoxidation are faster than wild-type (Table 1). The F28L mutant shows a 7-fold increase whereas the F28W substitution causes a larger, 14-fold increase. The highest autoxidation rate is observed for the F28H mutant, showing a 50-fold increase vs WT (Table 1). The large differences observed in the F28H are unexpected given the aromatic character of both side chains. The His side chain may adopt a different conformation to that of the native Phe residue, and can also interact with a water molecules. In the absence of structural data, the true cause of this divergent behavior remains unclear. The ability of phenylalanine to stabilize the Fe^II-O₂ complex has been shown in other studies of Mb and plant hemoglobins ^{18, 20}. In our case, the rate increase does not seem to relate to the size of the side chain as observed for sperm whale myoglobin ¹⁸. Our results are more comparable to the studies of the B10 residue in plant hemoglobins, where a phenylalanine was also the only residue providing stable oxygen binding ²⁰. Similar results have been also reported in myoglobin, where a phenylalanine in position B10 yields the lower autoxidation rates, albeit the reactions are several orders of magnitude slower to those seen in Ngb ¹⁸.

Figure 3. Autoxidation of Ngb Phe28 mutants.

The plots show selected spectra during the time course of autoxidation for each mutant. Arrows indicate the direction of the absorbance change. Insets show the fitting of the decay to a single exponential equation. A; F28V; B, F28H; C, F28W, D, F28L.

The changes in the Val68 (E11) position modified the reactivity towards oxygen in opposite directions. The V68A mutant did not form a stable oxy complex, and as observed in the Phe28 mutants a fast decay through an outer sphere mechanism is observed (Figure 4A). Conversely, replacement of Val68 by Phe or Ile (Figures 4B & 4C) resulted in mutants showing near-complete formation of a Fe^II-O₂ species. These complexes decay at a rate 2 to 6-fold lower than these of the wild-type oxy complex (Table 1).

Figure 4. Autoxidation of Ngb Val68 mutants.

The plots show selected spectra during the time course of autoxidation for each mutant. Arrows indicate the direction of the absorbance change. Insets show the fitting of the decay to a single exponential equation. A; V68A; B, V68I; C, V68F.

Nitrite reduction by mutant neuroglobins

Neuroglobin, as other heme proteins, can catalyze the reduction of nitrite to form NO according to the scheme:

$${\text{Ngb-Fe}}^{\text{II}}+{{\text{NO}}_2}^{-}+{\mathrm{H}}^{+}\to {\text{Ngb-Fe}}^{\text{III}}+{\text{NO}}^{•}+{\text{OH}}^{-}$$ (reaction 1)

$${\text{Ngb-Fe}}^{\text{II}}+{\text{NO}}^{•}\to {\text{Ngb-Fe}}^{\text{II}}\text{-NO}$$ (reaction 2)

As reaction 2 (rate constant around 2×10⁸ M⁻¹s^{−1 25}) is much faster than reaction 1, the reaction 1 is rate-limiting. It should be noted that in six-coordinate globins the binding of diatomic ligands is in practice limited by the dissociation rate of the distal histidine (~0.4 s⁻¹ for Ngb at 20 °C ³⁰); however the observed rates for Ngb mutants that retain the His64 residue are well below these values. In the presence of excess dithionite, the ferric Ngb formed is reduced back to ferrous Ngb; and the global process is as follows:

$${\text{Ngb-Fe}}^{\text{II}}+{{\text{NO}}_2}^{-}+{\mathrm{H}}^{+}{\to }^{-}{\text{Ngb-Fe}}^{\text{II}}\text{-NO}+\text{OH}$$ (reaction 3)

Removal of the distal histidine residue produces a five-coordinate heme that shows increased affinity towards ligands ^{4, 25, 26}. We have shown that the replacement of His64 by Gln or Leu leads to large increases in nitrite reduction rates ⁴. Here, we wanted to expand these studies by investigating the effect of other mutations of very different physicochemical properties. The observed spectral changes and calculated rate constants are shown in Table 2 and Supplemental Figures 1–4.

Table 2.

Nitrite reduction rates of wild-type and mutant deoxy-neuroglobins.

Ngb mutation	k Nitrite (M−1s−1)
Wild-type	0.52 ± 0.19
F28W	0.76 ± 0.24
F28L	2.70 ± 0.92
F28V	2.24 ± 0.33
F28H	0.37 ± 0.05
H64W	7.6 ± 1.3
H64Q	285 ± 23
H64A	1120 ± 140
V68A	0.090 ± 0.019
V68F	1.17 ± 0.34
V68I	5.07 ± 1.83

The mutation H64W retains the aromatic properties of the side chain and is theoretically able to form a hydrogen bond with the heme, albeit the larger size may cause conformational challenges for such interaction. The H64A mutation eliminates the ability to form hydrogen bonds and decreases side chain size. The H64W mutation increases nitrite reduction rates by 15-fold (Table 2). Replacement of His64 by Gln leads to a 700-fold increase, and in the case of H64A a rate over 2000-fold higher is observed. These changes are consistent with a marked dependence on the size of the side chain in position E7 (Table 2).

F28 mutants show a modest effect in their nitrite reductase rates, with values from 0.7 to 5-fold changes in the nitrite reduction rate as compared to wt Ngb. F28W and F28H mutations retain the aromatic character of the side chain and show the smaller changes. Replacement of the phenylalanine residue for a hydrophobic but not aromatic side chain (F28L and F28V) leads to 4–5 fold rate increases. These results suggest that the size of the side chain is the main factor in the rate.

In the case of the Val68 (E11) position, we observe again different trends between the V68A and V68F/V68I mutations. The V68A mutant reduced nitrite at a rate 6-fold slower than wild-type Ngb, whereas the other mutations cause rate increases of 2-fold (V68F) or 10-fold (V68I). We do not appreciate a direct effect of the side chain volume in the nitrite reduction rates as we see in Phe28 or His64; it is possible that significant rearrangements in the heme pocket are required to accommodate the side chain changes. In particular, the results observed for the V68A mutant suggest that some degree of collapse of the heme pocket may occur.

Redox potential of mutant neuroglobins

In order to study the effect of the redox potential on the kinetic parameters, we determined the redox potential of the neuroglobin wild-type and mutants at 25 °C in 100 mM Sodium phosphate, 100 mM, pH 7.0. (Table 3, Supplemental Figures 5–8)

Table 3.

Redox potentials of wild-type and mutant neuroglobins.

Ngb mutation	Em (mV)
Wild-type	−118 ± 4
F28W	−136 ± 4
F28L	−141 ± 4
F28V	−146 ± 5
F28H	−187 ± 2
H64W	−53 ± 22
H64Q	−62 ± 6
H64A	−119 ± 2
V68A	−107 ± 11
V68F	−121 ± 3
V68I	−126 ± 11

We first determined the redox potential of our wild type Ngb (Figure 5A, Table 3). Our observed value of -118 ± 4 mV is in good agreement with the reported values of −129 mV ²⁶ and −115 mV ²¹.

Figure 5. Redox potential determination for Ngb wild-type and mutants.

The plots show the fit of the fraction reduced (as determined from absorbance spectra) to the Nernst equation (solid lines). The titration of wild-type Ngb is included in all panels for reference. Panel A, His64 mutants; Panel B, Phe28 mutants; Panel C, Val68 mutants.

Mutation of the distal histidine either had no effect on the redox potential or shifted the midpoint potential to more positive values (Figure 5A, Table 3). The H64A mutation showed little effect, whereas H64Q and H64W caused increases of +56 and +65 mV, respectively. The redox potential for the His(E7)Leu mutation in several six coordinate globins has been studied by Halder et al.²¹. They observed a large increase in redox potentials for the mutation in rHb1, Cgb and SynHb, between +113 and +37, but the potential of Ngb H64L was almost unchanged ²¹. We observe a similar phenomenon with H64A, where the potential is similar to wild-type Ngb. However, H64Q and H64W are more positive as expected. In terms of water coordination we do not observe a correlation with the observed potentials. The ferric form of H64Q is clearly five coordinated, whereas the spectra of the H64A and H64W ferric forms are consistent with a mainly water-bound heme (Supplemental Figure 7).

The presence of a bound water molecule to the ferrous heme has been shown to correlate with a more negative redox potential in myoglobin His64 (E7) mutants ²². In our study, H64A retains the double peak of the ferrous form, whereas H64Q and H64W form a single peak ferrous species. The trend in redox potential appears to match the observations in Mb, with H64Q and H64W showing a shift towards more positive values. Similar observations have arisen from the study of His(E7)Leu mutants in six-coordinate globins, where the mutations in Ngb and SynHb retain a six-coordinate character and show less change redox potential than the corresponding mutations in rHb1 or Cgb where the mutant shows a five coordinate ferrous spectra ²¹.

Replacement of the Phe28 residue caused in all cases a decrease in the redox potential of the heme. The shift was moderate for F28W, F28L and F28V mutants (−18 to −28 mV, Figure 5B, Table 3) and more pronounced for F28H (−69 mV, Table 3)). Subtle structural changes may happen in the F28H mutant, as discussed in previous sections. The trend observed is similar to that reported in a study of another six-coordinate globin, rice non-symbiotic hemoglobin 1 (rHb1). In rHb1, the replacement of the B10 Phe residue by Trp or Leu also induced a decrease in the redox potential by 8 and 30 mV respectively ²⁰.

The mutation of the Val68 (E11) residue induced a much smaller shift in the redox potential, as compared to the effect of mutations on Phe28 (B10) or His64 (E7). The values of V68A, V68F and V68I are within ±11mV of the wild-type value (Figure 5C, Table 3). Mutation of the homologous residue in myoglobin by polar residues can produce redox potential decreases of more than 180 mV ³¹. As the studied mutation retain the hydrophobicity of the wild-type residue, the possible effect of such polar side chains on the redox potential of six-coordinate globins remains uncertain.

DISCUSSION

The mutation of the distal heme pocket residues in neuroglobin offers an opportunity to compare the properties of five-coordinate and six-coordinate heme globins. An extensive literature of protein engineering studies of five-coordinate globins exists. However, the applicability of these studies to six-coordinate globins is unknown. Here we show a systematic study of Ngb focusing on the effects on nitrite reductase rates, autoxidation kinetics and redox potentials.

Nitrite reductase rates and redox potential

Previous studies of the nitrite reductase reaction on Hb and Mb showed a trend where the rate of the reaction was faster at more negative heme potentials ¹⁴. Subsequent work on six-coordinated globins does not seem to conform to this pattern. Whereas six coordinate globins have in general lower redox potentials than five-coordinate globins ^{21, 26}, their nitrite reduction rates are not consistently faster. Ngb and Cgb appear to be slower nitrite reductases than Mb ^{4, 17, 32} and six-coordinate plant hemoglobins are faster nitrite reductases than Mb ^{15, 16}.

The rates of nitrite reduction by Ngb mutants can be plotted versus their heme redox potential (Figure 6). There is no clear relationship between redox potential and nitrite reductase activity. The more striking feature of the plot is the fact that His64 (E7) mutations greatly increase the nitrite reduction rate, independently of the redox potential of the mutant (Figure 6).

Figure 6. Relationship between the observed nitrite reductase rates and the redox potential.

The different symbols denote different Ngb mutations as follows: solid square, Ngb wild-type; solid circles, Phe28 mutants; up triangles, His64 mutants; down triangles, Val68 mutants; empty circle, wild-type sperm whale Myoglobin; solid circle, human Hemoglobin. The dashed lines indicate the breaks in the X and Y axes.

In general, the nitrite reduction in Ngb appears to be limited by heme iron accessibility, and this limitation is partly contributed by the heme distal pocket but mainly depends on the His (E7) ligation to the heme. In this context, it is tempting to speculate that the rate of nitrite reduction in six-coordinate globins will correlate with the distal histidine binding affinity. However, the relationship between the two factors appears to be more complex. A summary of histidine binding parameters for six-coordinate globins is shown in Table 4. As pointed out by Sturms et al.¹⁶, tight binding of distal histidine (K_His>100) can be observed in slow nitrite reductases, such as wild-type Ngb, but also in fast nitrite reductases, such as SynHb. Conversely, weaker distal histidine binding (K_His<100) is observed in Cgb (slower reductase) and rHb1 (fast reductase). It is noteworthy that Ngb and Cgb have the slower histidine dissociation rates (Table 4). Actually, histidine dissociation rates are an imperfect, yet better indicator of the nitrite reduction rates. Although the concentrations of nitrite used do not seem high enough to overcome the fast histidine binding rates, it is possible that the presence of a nitrite in the heme pocket increases histidine dissociation rates by forming electrostatic and H-bonding interactions with the distal histidine side chain. Different reaction mechanisms for five- and six-coordinate globins may exist; this possibility is discussed below.

Table 4.

Distal histidine binding constants, nitrite reductase rate constants and redox potentials for selected globins.

Protein	Distal histidine binding			KNitrite (M−1s−1)	Em (mV)
	kbinding (s−1)	kdissoc (s−)	KHis
Ngb (human)	2000 a	4.5 a	444 a	0.12 b	−118 c
Cgb (human)	200 d	2 d	100 d	0.14 e	−28 f
SynHb	4200 g	14 g	300 g	27 h	−195 f
RHb1	75 g	40 g	1.9 g	40 h	−143 f
AtHb1	230 i	110 i	2.1 i	19.8 j	ND
AtHb2	1600 i	38 i	42 i	4.9 j	ND
SwMb	NA	NA	NA	5.6 b	−59 k

Histidine dissociation determined at 20 °C in 100 mM sodium phosphate buffer, pH 7.0. Nitrite reductase rates determined at 25 °C in 100 mM sodium phosphate buffer, pH 7.4 except Cgb, SynHb, and RHb1, determined at pH 7.0. All redox potentials determined at 25 °C in 100 mM sodium phosphate buffer, pH 7.0. Values from

^a26;

^b4;

^cThis work;

^d45;

^e4;

^f21;

^g46;

^h16;

ⁱ⁴⁷;

^j15;

^k31.

NA, not applicable. ND, not determined.

Autoxidation rates and redox potential

We have investigated the relationship between the observed autoxidation rates for Ngb mutants and the redox potential of the heme group (Figure 7). In this case, we can observe a reasonable correlation between the autoxidation rates and the redox potential. The mechanisms of globin autoxidation have been discussed in detail by Shikama ^{33, 34}. The potential for formation of superoxide from oxygen is −330 mV; thus theoretically a heme potential of −330 mV would be needed to provide the driving force necessary for the reaction to occur spontaneously ^{33, 34}. It follows that an efficient oxygen carrier protein will experience a evolutionary pressure to minimize this side reaction, and consequently proteins like Hb and Mb have a significantly positive reduction potentials(+59 mV for Mb ³¹; +150 mV for Hb ³⁵). In general, lower redox potentials have been determined for Ngb (−118 mV, Table 3) and other six-coordinate globins ²¹. In a hypothetical scenario where the redox potential constitutes the sole driving force of the reaction, the more negative the heme redox potential the faster the heme autoxidation (and concomitant oxygen reduction) will be. Our results fit well into this overall scheme, with the proteins with more negative potential showing faster autoxidation rates (Figure 7). The inset of Figure 7 is consistent with a change in the activation energy of the reaction that depends linearly of the redox potential, as dictated by the Arrhenius equation. It is remarkable that this observation requires that all other factors (including, but not limited to ligand accessibility and pH effects) have a limited influence in the rates. We have also included in the analysis a subset of four E7 Mb mutants for which the redox potential and autoxidation rates are available (Supplemental Table 1) ^{18, 22}. We do not observe a consistent behavior for the mutant proteins and the wild-type Mb; however, a separate analysis of the mutants alone yields a linear fit, almost parallel to the neuroglobin data (Figure 7). This indicates that for some Mb mutants a situation similar to Ngb, with the redox potential dominating the rate, is plausible when the distal pocket is enlarged and does not provide much ligand stabilization. It is unlikely that this situation can be generalized to other Mb mutants, as the redox potential of the Mb mutants is not far from wild-type Mb values and most mutations do not show such large deviations from wild-type Mb autoxidation rates as the E7 mutants (Table 5).

Figure 7. Relationship between the observed autoxidation rates and the redox potential.

The different symbols denote different Ngb mutations as follows: solid square, Ngb wild-type; solid circles, Phe28 mutants; up triangles, His64 mutants; down triangles, Val68 mutants; empty circle, wild-type sperm whale Myoglobin; solid circle, human Hemoglobin. The empty squares denote sperm whale Myoglobin mutants; mutations are indicated in italics. The inset shows the same points but using a logarithmic scale for the autoxidation rates; the solid line denotes the best fit of the Neuroglobin data points to a linear equation; the dotted line denotes the best fit of the sperm whale Myoglobin data points (except wild-type) to a linear equation.

Table 5.

Autoxidation rates for neuroglobin and myoglobin mutants.

Ngb mutation	kautox a (min−1)	swMb equivalent	kautox b (h−1)
Wild-type	0.23	L29F	0.005
F28W	3.22	L29W	ND
F28L	1.76	Wild-type	0.051
F28V	3.06	L29V	0.23
F28H	11.3	L29H	ND
F28A	ND	L29A	0.24
H64W	0.076	H64W	ND
H64Q	0.010	H64Q	0.21
H64A	0.066	H64A	58
H64L	ND	H64L	10
H64G	ND	H64G	44
H64V	ND	H64V	33
H64T	ND	H64T	54
H64F	ND	H64F	6
V68A	2.38	V68A	0.26
V68F	0.039	V68F	0.069
V68I	0.122	V68I	0.75
V68L	ND	V68L	0.10

^aValues determined at 37 °C in 100 mM sodium phosphate buffer, pH 7.4 (this work).

^bValues determined at 37 °C in 100 mM potassium phosphate buffer, pH 7.0 ¹⁸.

ND, not determined.

Effects of mutations on Ngb versus Mb

Autoxidation rates of a variety of Mb mutants are available ¹⁸. Notwithstanding the heterogeneity within five-coordinate globins ³⁶, this offers a reasonable framework to compare the effect of mutations on five and six-coordinate globins. A comparison of the observed values is shown in Table 5.

Mutations of the B10 residue (Phe28 in Ngb, Leu 29 in swMb) indicate a common pattern for both globins. In both cases the phenylalanine provides the maximal stability for the Fe^II-O₂ complex (Table 5). Studies on the six-coordinate rHb1 have also indicated that a phenylalanine in position B10 is also the residue allowing for a slower autoxidation rate ²⁰. The conservation of this residue in Ngb sequences can be related to a role of the protein in O₂ transport/storage, but other functions such as NO scavenging would also benefit from a stable oxy species ²⁰. In any case, we observe that other mutations can also increase Fe^II-O₂ complex stability in Ngb, notably His64 substitutions (Tables 1 & 5). However, His64 and Val68 (and Phe28) are strictly conserved in the Ngb sequences available. This observation suggests that the O₂ complex stability of wild-type Ngb is enough for its biological function, and other factors such as six-coordination are apparently more relevant to function. Remarkably, not all six-coordinate globins conserve a Phe residue in position B10 (for example a leucine residue is conserved in Cgbs); this suggests that the stability of the Fe^II-O₂ species has not been optimized during evolution of six-coordinate globins.

Mutations of the residue in position E11 had a smaller effect on Ngb autoxidation rates than the equivalent mutations in swMb (Table 5). The Ngb V68A mutation was an exception to this trend, but based on the slow nitrite reduction by this mutant we hypothesize that substantial rearrangements of the heme environment, with a reduction of the accessible volume of the heme pocket, occur in this mutant.

Given the particular role of the E7 residue in six coordinate globins, is not completely unexpected that the differences between Ngb and Mb E7 mutants are maximal. Whereas this mutation increases autoxidation up to 1000-fold in Mb, all the Ngb mutants show improved Fe^II-O₂ stability. The increase in autoxidation rates is not completely general to five-coordinate globins; in fact the His(E7)Leu mutant of soybean leghemoglobin shows only a modest increase in autoxidation ³⁶. In Mb, the E7 histidine is both stabilizing bound O₂ and preventing its protonation (which leads to fast autoxidation via the formation of Fe^II-O₂H+ and fast dissociation to Fe^III and HO₂). This stabilized FeII-O₂ species is reportedly inert towards autoxidation ¹⁸. The CO-bound Ngb structure shows the His side chain close to the CO molecule ³⁷, and it is thus conceivable that the His E7 in Ngb could fulfill a similar role. However, our observed rates for His(E7) mutants indicate that a histidine is not necessary for the stabilization of the Fe^II-O₂ species in Ngb. The differences between Ngb and Mb autoxidation reactions could also be mechanistic. Two different mechanisms (unimolecular versus bimolecular) can be involved in the autoxidation reaction ¹⁸. The swMb data shows a reasonable correlation between autoxidation rates and oxygen dissociation constants; the correlation is consistent with a unimolecular autoxidation mechanism being predominant at higher oxygen concentrations ¹⁸. We investigated the nature of the autoxidation reaction mechanism in Ngb (Supplemental Figure 9). Our experiments indicate that wild-type Ngb and the F28W mutant show rates consistent with an unimolecular mechanism with little or no contribution from the bimolecular mechanism, unlike the mixed mechanism generally observed in swMbs ¹⁸. In the case of the H64A mutant, a behavior more consistent with a mixture of uni- and bimolecular mechanism is observed. The contribution of a bimolecular autoxidation mechanism to the observed rate declines as the oxygen concentration increases, and approaches zero at high [O₂], whereas the unimolecular mechanism rate increases in a hyperbolic manner reaching a saturation rate. As the autoxidation rates seem to reach a plateau at oxygen levels higher than 50 μM for the Ngbs studied, we expect the unimolecular mechanism to prevail in normoxic conditions for Ngb as it does for Mb. Therefore, a reasonable explanation for the opposite effect observed for the autoxidation rates of E7 mutants in Mb and Ngb can be traced to the opposite changes in oxygen affinity. The E7 mutants of swMb show a decreased affinity towards oxygen ¹⁸, but the data on Ngb mutants H(E7)Q and H(E7)V indicates that the oxygen affinity is increased with respect to the wild-type protein ²⁹. We speculate that oxygen affinities for the Ngb mutants will also show a correlation with their autoxidation rates, indicating that the dissociation of oxygen or superoxide from the heme are regulated by similar constraints.

Two mechanisms of nitrite reduction?

Our results further indicate a dichotomy in the reaction of deoxy five- and six-coordinate globins towards nitrite. In Hb and Mb there is a correlation between faster rates and more negative potentials ¹⁴. However, in Ngb there is a marked increase in reaction rates as the distal pocket size increases, and redox potential shows a poor relationship with reaction rate constants. These observations are puzzling; if the reaction of nitrite were limited in all cases by heme pocket size, Hb and Mb should show faster rates than six-coordinate globins. Differences in reaction mechanism may exist; Perissinotti et al. studied the nitrite reaction with deoxyhemoglobin by computational methods and showed that histidine protonation could be a limiting step in the reaction ³⁸. Our studies showed that swMb mutants His(E7)Ala and His(E7)Leu are indeed poor nitrite reductases (Supplemental Table 1) ⁴. In the case of horse Mb, the His(E7)Val mutant causes a 15-fold decrease in nitrite reduction rate ³⁹. Conversely, our results indicates that for Ngb the reduction mechanism does not rely on proton delivery from the His side chain, as His64 mutants show the fastest reduction rates. If the proton is delivered from a water molecule, we would expect that mutants such as F28H or H64Q could help to keep that water molecule in place, but they are not faster than F28L or H64A, respectively.

The reaction of nitrite with deoxy globins requires the formation of an elusive Fe^II-NO₂- intermediate ⁴⁰. The geometry of the intermediate is a matter of discussion, as the nitrite molecule can be bound through the nitrogen atom (Fe^II-N-nitro), one oxygen atom (Fe^II-O-nitrito) or even a bidentate (Fe^II-O,O-nitrito) species ^41–44. Interestingly, the product of a nitrogen-bound nitrite will be Fe^III-NO ^{4, 38}, whereas an oxygen-bound nitrite will yield a Fe^III-OH intermediate ³⁸. Recent studies by Silaghi-Dumitrescu et al. ⁴⁰ indicate that in the reaction of hemoglobin a Fe^III-NO intermediate species can be detected, therefore implying a Fe^II-N-nitro intermediate. It is conceivable that the subtle structural changes between five- and six-coordinate globins can lead to a difference in the stabilization of the Fe^II-NO₂- species. The possibility of five-coordinate globins favoring N-nitro binding modes and six-coordinate globins favoring a more reactive O-nitrito species deserves further study.

ABBREVIATIONS

Hb

red blood cell hemoglobin

Mb

myoglobin

Ngb

neuroglobin

Cgb

cytoglobin

rHb1

rice non-symbiotic hemoglobin 1

B10

tenth amino acid of the B helix

E7

seventh amino acid of the E helix

E11

eleventh amino acid of the E helix

K_His

equilibrium constant for the distal histidine binding equilibrium (rate of His binding divided by rate of His dissociation)

NO

nitric oxide

SHE

standard hydrogen electrode

SynHb

Synechocystis hemoglobin

swMb

sperm whale myoglobin