Rational Design of a Structural and Functional Nitric Oxide Reductase

Summary

Protein design provides an ultimate test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. While progress has been made in designing proteins that mimic native proteins structurally^1–3, it is more difficult to design functional proteins^4–8. In comparison to recent successes in designing non-metalloproteins^4,6,7,9,10, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes^5,8,11–20, since protein metal binding sites are much more varied than non-metal containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal binding site properties in silico, as many of the parameters for metal binding sites, such as force fields are ill-defined. Therefore, the successful design of a structural and functional metalloprotein will greatly advance the field of protein design and our understanding of enzymes. Here, we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a heme/non-heme Fe_B center that is remarkably similar to that in the crystal structure. This designed protein also exhibits NOR activity. This is the first designed protein that models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.

NOR is a metalloenzyme in the denitrification pathway of anaerobic bacteria that is responsible for the two electron reduction of NO to N₂O²¹. It is a key enzyme in the nitrogen cycle that is critical for all life. Furthermore, well-studied denitrification enzymes may provide structural and spectroscopic models for mammalian enzymes that produce and utilize NO in signal transduction pathways. Progress in understanding NOR has been hampered by difficulties in obtaining enzyme in high yield and the lack of a three-dimensional structure. Peptide sequence alignments and structural threading have suggested that NORs are structurally homologous to subunit I of heme-copper oxidases (HCOs)²². All six His ligands to the active site of HCO (two to the low spin heme, one to the high spin heme, and three to Cu_B) are conserved in NOR at the same predicted positions in HCO. The major structural difference is the replacement of a copper binding site (Cu_B) with a non-heme iron binding site (Fe_B) in NOR^21,23. Additionally, several conserved glutamates (not found in HCOs), have been identified near the catalytic heme/non-heme Fe_B site of NOR, and are predicted to play a role in iron binding and/or catalysis^24,25. Synthetic models of NOR have been reported^21,26,27. Despite these progresses, computational design and 3D structural confirmation of structural and functional models remain elusive, and it is often difficult to address long-range non-covalent interactions important for enzymatic functions using these models.

We chose to use sperm whale myoglobin (swMb) as a scaffold protein in which to engineer an NOR active site because it is much easier to prepare and crystallize than both native proteins and synthetic models of NOR. Myoglobin and its mutants are also more amenable to biophysical characterization due to the presence of a single metal center, free from interference by other chromophores. Although swMb contains a heme center, it lacks the Fe_B center and NO reduction activity of NOR.

To design an Fe_B center in swMb, we examined its distal pocket for locations to introduce the putative non-heme metal binding site, consisting of three histidines and one glutamate. The histidine residues were introduced first¹⁴, as crystal structures of HCOs containing three conserved histidines are readily available (e.g., CcO, cytochrome c oxidase). Based on overlays of the minimized structure of swMb and CcO, the distal His64 in swMb was chosen as one of the histidines, while Leu29 and Phe43 were mutated to histidines, to produce a non-heme metal binding site. The V68E mutation to swMb was chosen based on its proximity and angle to the heme and the three His. The minimized computer model of the resultant triple mutant (swMb L29H, F43H, V68E), named Fe_BMb, is shown in Fig. 1A. The Fe_B site (modeled as Zn(II)) is within bonding distance to Nε of all three His residues (2.13 Å to H29, 2.20 Å to H43, and 2.09 Å to H64) and both O atoms of E68 (2.11 Å and 1.96 Å), indicating that the proposed mutations would support iron binding in myoglobin, forming an Fe_B site. A comparison of the computer model overlaid with the crystal structure of native CcO (see Fig. S1A) suggests structural similarity between the two centers.

Figure 1.

Crystal structure of rationally designed Fe_BMb overlays closely with minimized computer model. A) Minimized computer model of Fe_BMb with Zn(II) in the Fe_B site. B) Crystal structure of Fe(II)-Fe_BMb collected at Fe-edge absorption (1.7309 Å) at the Brookhaven National Synchrotron Light Source X12C beamline (Upton, NY) with 1.72 Å resolution. The Fe•••Fe distance is 4.82 Å, while the Fe-O-Fe angle is 115° (OE1 atom of E68). C) Overlay of Fe_BMb model (yellow) with Fe(II)-Fe_BMb crystal structure (cyan). In general, Fe(II) of the Fe_B site is represented by a green sphere; Zn(II) (grey sphere) was used to model the Fe_B site. A water molecule in the heme pocket is represented by a red sphere.

To test designed Fe_BMb for iron binding, Fe²⁺ was titrated into deoxy (reduced heme iron) Fe_BMb that contains heme, but not any metal ions in the Fe_B site. The Soret band of deoxy Fe_BMb shifted slightly from 433 nm to 434 nm with Fe²⁺ addition. Concurrently, the peak at 557 nm was split into two shallow bands at 550 nm and 572 nm (Fig. 2Aa). This observation provided evidence that Fe_BMb is capable of incorporating iron into its designed metal binding site, an important step toward modeling the Fe_B site of NOR. In contrast, titration of Fe²⁺ into either wtMb (Fig. 2Ab) or a Mb derivative with the three His but no Glu (called Fe_BMb(-Glu), Fig. 2Ac) resulted in no changes to the UV-vis spectra, indicating that both the histidines and particularly the glutamate, are required for Fe²⁺ binding.

Figure 2.

Designed Fe_BMb has NOR activity in the presence of Fe²⁺ and NO. Control proteins show no evidence of NO activity under identical conditions. A) UV-vis spectra of 10 μM of deoxy Fe_BMb (a), deoxy wtMb (b), and deoxy Fe_BMb(-Glu) (c) in the absence (black spectra) or presence (red spectra) of 2 eq Fe²⁺. B) UV-vis spectra of the NO reaction of 10 μM of deoxy Fe_BMb (a), deoxy wtMb (b), and deoxy Fe_BMb(-Glu) (c) in the absence (black spectra) or presence (red spectra) of 2 eq Fe²⁺ after 20 min of incubation with ~17 eq NO. The deoxy protein was formed by reaction of met protein with excess dithionite in a glove box. Gel filtration purification was used to remove excess dithionite to preclude its reaction with NO.

Further evidence of Fe²⁺ binding to the designed Fe_B site comes from a high resolution crystal structure (1.72 Å) of Fe(II)-Fe_BMb (Fig. 1B, S1, S5, Table S1). The non-heme iron in the Fe_B site is five coordinate, with bonds to all three histidines (2.18 Å to H29, 2.12 Å to H43, and 2.20 Å to H64), one O atom of E68 (2.19 Å) and one water molecule (2.11 Å). A weaker interaction between the non-heme iron and the other O atom of E68 at 3.28 Å is also present. The crystal structure presented here is consistent with the proposal that a glutamate in the active site of NOR helps stabilize iron binding to the Fe_B site. In addition to structural roles, the glutamate, histidines and water in the active site may also provide the two protons required for NO reduction.

The overlay of the crystal structure of Fe(II)-Fe_BMb with the computer model (Fig. 1C) is striking. Although His43 does not overlay as well since it is located in a flexible loop, the other two His and Glu68 overlay well with one another. Zn(II) in the model and Fe(II) in the Fe_B site do not overlay exactly, but are in a similar position, 1.08 Å away from each other. Furthermore, the model did not predict the recruitment of a water molecule into the heme pocket, possibly because Zn(II) was used to model Fe(II), in the absence of Fe(II) parameters. Future development of Fe(II) parameters (not in a heme) will likely help resolve this issue. The heme in the computer model is puckered instead of planar, causing the iron to be pulled ~ 1 Å out of the heme plane. If out-of-plane distortion of the model is considered, the Fe_B to heme iron distance is ~ 4.8 Å for both the model and crystal structure. Therefore, the distinct similarities between the crystal structure and computer model suggest that metalloprotein active sites can be rationally designed.

Since the Fe_B iron is only ~ 4.8 Å away from the heme iron, it is possible for spin-coupling to occur between the two irons, as is shown in native NOR^23,28. To find out if the designed Fe_BMb mimics this aspect of native NOR, we titrated different equivalents of Fe²⁺ into deoxy Fe_BMb and then added blue copper Azurin as a redox partner. In the absence of non-heme Fe²⁺ and Azurin, deoxy Fe_BMb displayed no EPR signal, as Fe(II) heme has an EPR-inactive integer electron spin in the perpendicular mode of EPR. Upon addition of Cu(II)-Azurin, a high spin Fe(III) heme EPR signal centered at g~6 was observed (Fig. S2), suggesting that Azurin can serve as a redox partner of Fe_BMb and oxidize the Fe(II) heme to Fe(III) heme. Interestingly, the intensity of the high spin heme signal decreased with Fe²⁺ binding to the Fe_B site of Fe_BMb, which suggests that Fe²⁺, when oxidized by Azurin, is spin-coupled to the Fe(III) heme, similar to that observed in native NOR. This conclusion is also supported by electrochemical redox potential determination, as the presence of iron in the Fe_B site resulted in a dramatic increase of the heme reduction potential (from −158 ± 4 mV to −46 ± 2 mV vs. NHE; Fig. S3).

To test its NO activity, deoxy Fe_BMb was reacted with excess NO (~17 eq) in the presence of Fe²⁺ and monitored by UV-vis (Fig. 2B). These conditions approximate one turnover as only two electrons are available for reaction, one from reduced heme iron and one from ferrous Fe_B. Furthermore, the reduction of NO to N₂O is highly thermodynamically favorable (E₀′ = + 1.2 V)²⁹, such that many common water soluble reductants can reduce NO non-enzymatically. Studying NO activity under single turnover conditions precludes non-enzymatic NO reduction and allows the reaction to be easily followed by spectroscopy. The appearance of visible peaks at 546 nm and 582 nm ~ 1 s after NO addition indicate fast NO binding. However, the NO-bound protein is not stable and a species with peaks at 408 nm, 550 nm and 577 nm forms immediately after NO binding and is stable with no other changes in the spectrum over the course of 20 min (Fig. 2Ba, red spectrum). The Soret band at 408 nm is characteristic of Fe(III) heme and the visible peaks are characteristic of an NO-bound heme, similar to the NO-bound oxidized Fe(III) heme or met form of Fe_BMb (Fig. S4, black spectrum). Therefore, it appears that NO addition to Fe(II)-Fe_BMb results in a single species, namely the met-NO form. In contrast, when the Fe_B site of Fe_BMb is empty, NO addition results in NO binding with no additional changes in the UV-vis spectrum over 20 min (Fig. 2Ba, black spectrum). Therefore, these observations provide evidence that Fe_BMb with an intact Fe_B site can react with NO, whereas Fe_BMb with a vacant Fe_B site binds, but does not react with NO.

As further controls, deoxy wtMb lacking an Fe_B site, and an Fe_BMb derivative lacking the glutamate (Fe_BMb(-Glu)) were reacted with NO in the presence or absence of Fe²⁺. After 20 min incubation with NO, the UV-vis spectra of both proteins in the presence of Fe²⁺ remains unchanged (Fig. 2Bb and Bc), suggesting the lack of NO activity for both proteins. These results are not surprising since Fe²⁺ binding studies (Fig. 2Ab and 2Ac) indicate no evidence of iron binding for either proteins. Therefore, the histidines and glutamate are important not only for iron binding, but also for NOR activity.

The product of NO reduction was identified by GC/MS under single turnover conditions by monitoring NO formation in the headspace of the solution. Because of the high solubility of N₂O (~ 25 mM in water at room temperature), this method cannot be used to quantify the rates of the NO reduction under 1–2 turnover conditions (see Supplemental Information, Fig. S8). Typical MS of NO (MW 30) and N₂O (MW 44) are shown in Fig. S6. When Fe(II)-Fe_BMb was incubated with NO, N₂O production was observed by the appearance of a second peak in the GC with a longer retention time than NO (Fig. 3A). Although the amount of N₂O formed by Fe(II)-Fe_BMb is low, it is consistently more than that produced by Fe²⁺ alone (without protein, Fig. 3B) over multiple samples. After no additional changes were observed over a period of 1–2 hr, 2 eq of dithionite was added at 6 hr to reduce the heme and Fe_B sites again, approximating a second turnover. Although considerable N₂O was produced in both samples (dithionite can non-enzymatically reduce NO to N₂O), Fe(II)-Fe_BMb produced ~ 2 times more N₂O than the control sample without protein, as observed in the chromatogram of only the 44 MW component, representing the main ion peak of N₂O (Fig. S7, bottom blue traces). As an additional control, wtMb in the presence of Fe²⁺ was reacted with NO with no N₂O formation observed (Fig. 3C), indicating that non-specifically bound iron, if present, does not contribute to NOR activity. The yield of N₂O production by Fe(II)-Fe_BMb was estimated to be ~ 30%. These results, together with UV-vis data (Fig. 2), indicate that designed Fe_BMb is a functional model of NOR.

Figure 3.

Product of NO reaction by Fe_BMb is N₂O. Time dependent GC/MS measurements of N₂O formation by Fe(II)-Fe_BMb (A). Control reactions of NO with Fe²⁺ only (B) and wtMb (C). NO (~17 eq) was reacted with Fe(II)-Fe_BMb (0.6 mM protein, 1.5 mM or 2.5 eq Fe²⁺), Fe²⁺ (1.5 mM, no protein), or with wtMb (1 mM) and Fe²⁺ (2 mM or 2 eq). At 6 hr, 2 eq dithionite (1.2 mM) was added and allowed to react to simulate a second turnover. NO₂ (MW 46) was not detected. GC peaks have been normalized. N₂O yield (30%) was determined from a comparison of the ratio of the NO:N₂O peaks of the 30 MW:44 MW single ion chromatograms 2 hr after dithionite addition (after which additional N₂O formation was not observed), to that of known dithionite concentrations (2 hr after addition). Background N₂O formation (i.e., Fe²⁺ without protein) was subtracted from that of Fe(II)-Fe_BMb to estimate the yield.

We have rationally and successfully designed a protein model that both structurally and functionally mimics native NOR. We have demonstrated that both the glutamate and histidine ligands, which are conserved in NOR, are essential for iron binding and NOR activity. Being much smaller, easier to express in high yields, and free of other chromophores, this designed NOR protein will serve as an excellent model for mechanistic studies of NOR.

METHODS

Fe_BMb computer modeling

A computer model of the designed protein Fe_BMb was constructed with the L29H, F43H, and V68E mutations to the crystal structure of swMb (PDB: 1JP6) using the modeling extension in VMD (Visual Molecular Dynamics)³¹. Rendering was also performed in VMD. H43 was rotated about Cα to ensure interaction with the heme pocket as opposed to the bulk solvent. To assess the ability of the designed Fe_B site to bind an iron ion, molecular dynamics simulations were performed. To mimic an iron ion, the water molecule found in crystal structure of the distal pocket of swMb, was changed to a Zn(II) ion. The resulting computer model was then minimized with NAMD (Molecular Dynamics Simulator)³² using 5000 minimization steps at 0 K, then 10,000 molecular dynamics steps (1 fs/step) via an NVT ensemble at 310 K.

Construction, expression and purification of Fe_BMb

The triple mutant L29H/F43H/H64/V68E of sperm whale myoglobin (called Fe_BMb) was constructed using a protocol described previously^14,33 and confirmed by DNA sequencing at the Biotechnology Center of the University of Illinois. The DNA was transformed into BL-21(DE3) cells, expressed overnight, and harvested by centrifugation. The bacterial pellet from the equivalent of a 1 L growth was resuspended in 100 mL of 10 mM Tris (2-amino-2-hydroxymethyl-propane-1,3-diol) pH 8 containing 1 mM EDTA, 0.5 mM DTT (dithiothreitol), 35 units/mL DNaseI, 4 units/mL RNaseI, 200 mg lysozyme and a few crystals of PMSF (phenylmethylsulphonyl fluoride). The resultant solution was stirred at 4 °C overnight, diluted to 200 mL with the resuspension buffer, and centrifuged at 10,000xg for 45 min. The resulting grey/white pellet was resuspended in 50 mL of 7 M Guanidine HCl (GuHCl) and shaken at 4 °C for ~ 1 hr. A solution of 25 mg KCN/7.5 mg hemin in 5 mL H₂O was prepared and diluted by rapid mixing into 450 mL of cold H₂O. Heme incorporation was achieved by dropwise addition of the protein/GuHCl to the hemin-CN solution over 30 min with rapid stirring at 4 °C. The resulting cloudy red solution was stirred for ~ 1 hr after the final addition and centrifuged for 30 min at 8000xg to remove precipitate. The supernatant (500 mL) was dialyzed against 8 L of H₂O and 4 L of 100 mM (ionic strength) potassium phosphate (Kpi) at pH 7 to remove excess GuHCl. The protein was refolded with three additional buffer changes at least 6 hr between changes. The protein was then centrifuged for 30 min at 8000xg and concentrated with a stirred cell concentrator (8000 MWCO membrane; Millipore) to 10 mL. The concentrated protein was then loaded onto a gel filtration column (Sephacryl S-100 High Resolution; GE Healthcare) equilibrated with 100 mM Kpi pH 7 and eluted with the same buffer. The fractions with high (≥4.5) R/Z (A_406nm/A_280nm) were pooled, concentrated, and dialyzed twice against 100 mM Kpi pH 7 containing 30 mM EDTA (ethylenediaminetetraacetic acid), then twice with 100 mM Kpi pH 7 treated with a chelating resin (Chelex 100, iminodiacetic acid, Sigma) containing 100 mM NaCl, and finally three times with 100 mM Kpi pH 7 treated with chelating resin to remove adventitious metal. The purity and identity of purified Fe_BMb was confirmed by SDS-PAGE and ESI-MS (cal’d MW=17375 Da; exp’l MW= 17376+/−1 Da). Yield: ~ 13 mg protein/L of BL-21(DE3) bacterial growth.

UV-Vis characterization of Fe_BMb under one turnover conditions

UV-vis spectra were collected on either a Cary 3E (Varian) spectrometer connected to a circulating water bath equipped with a digital temperature controller (Polyscience), or a Cary 5000 (Varian) spectrometer equipped with a Quantum Northwest peltier temperature controller and stirring module. Kinetic UV-vis data were also collected to probe the reaction of NO with Fe_BMb in the absence or presence of Fe²⁺ using a Hewlett-Packard 8453 spectrometer with the kinetics module. A protein solution was degassed and transferred to a glove box where it was reduced, purified by size exclusion column, and reconstituted with 2 molar equivalents of Fe²⁺ (FeCl₂, Alfa Aesar) when required.

EPR characterization of spin-coupling of Fe_BMb in the presence of Fe²⁺

To prepare EPR samples, Fe_BMb was first reduced by dithionite in a glove box to form deoxy Fe_BMb. Excess dithionite was removed with a size exclusion column. Different molar equivalents of Fe²⁺ were then titrated (i.e., 0, 0.5, 1.0, 1.5, 2.0 eq) into the deoxy Fe_BMb solution. After stirring 20 min, 3 molar equivalents of oxidized wt Cu(II)-Azurin was added to each sample to oxidize Fe_BMb as well as the Fe(II) (Fig. S2). After 5 min, the sample was then transferred into an EPR tube and flash frozen in liquid N₂. EPR samples contained 0.5 mM Fe_BMb, 1.5 mM Azurin and 6.8% glycerol in 50 mM Bis-Tris (bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane) pH 7.0. Control experiments of wtswMb only, Fe_BMb(-Glu) only and Fe²⁺ only were prepared under identical conditions. Spectra were recorded at 4 K on a Bruker ESP 300 equipped with an Oxford liquid helium cryostat and an ITC4 temperature controller.

Spectroelectrochemical determination of reduction potential

Protein reduction potentials were measured using an optically transparent thin layer electrode (OTTLE) as previously described^30,34, with the following changes. A 200 μM protein solution, ~0.6 μL, containing 2 molar equivalents of Fe²⁺ when required, and 100 mM NaCl in 50 mM Bis Tris pH 7 buffer or 100 mM Kpi pH 7 buffer without added NaCl were used. Each protein solution also contained 80 μM each of phenazine methosulfate (PMS, Sigma) and anthraquinone-2-sulfonate (AQS, Sigma) as electron transfer mediators. For the spectroelectrochemistry study of deoxy Fe_BMb in the presence of Fe²⁺ (starting from the reduced deoxy metal bound form and transitioning to the oxidized met form), the protein was reduced in the glove box first with dithionite and purified of excess dithionite before mediator (AQS only) and Fe²⁺ addition, and subsequent transfer to the degassed OTTLE cell. The headspace of the OTTLE cell was purged with N₂ during the experiment to prevent O₂ contamination.

The potential of the working electrode was applied in the negative direction for deoxy Fe_BMb and in the positive direction for deoxy Fe_BMb + Fe²⁺. The UV-vis spectrum of the protein at each potential was recorded with a Cary 3E or Cary 5000 spectrophotometer until no further spectral changes were observed.

Data analysis

The spectra from 300–800 nm were analyzed by global analysis using singular value decomposition (SVD) and nonlinear regression modeling software (SpecFit/32, Spectrum Software Associates, Inc). The reduction potential of each protein solution was obtained by fitting the data to the best fit model, an A ↔ B model, in SpecFit/32 and checked by generating a Nernst plot at a single wavelength³⁰. The Ag/AgCl (3 M KCl) reduction potentials were converted to NHE potentials by adding 0.209 V. Since the reduction potentials obtained from the Nernst plots and from SpecFit/32 modeling were generally within ± 16 mV of each other, only the potentials derived from SpecFit/32 were reported (Fig. S3).

Purification of NO and NO saturated solution preparation

NO gas (Matheson Trigas, 99%) was purged of N₂O₃, NO₂, and higher N_xO_y impurities by passing it through a bubbler containing 1 M NaOH and a second bubbler of water before it entered a reaction vessel filled with ultrapure water³⁵. The reaction solution was first purged with helium to remove O₂ to make a ~1.1 mM saturated NO solution, as measured by spectroscopic titrations of a saturated solution of NO to oxy wtMb to form met wtMb. NO was added via a gas-tight syringe to a protein and/or metal solution.

X-ray crystallographic studies

Fe²⁺ incorporated Fe_BMb was prepared as above. Fe_BMb crystals were grown anaerobically in a glove box at room temperature. Red crystals were observed after 2–3 days. Diffractable crystals were obtained from a 1.5 mM protein solution in 20 mM Kpi pH 7 mixed 1:1 with well buffer (0.2 M sodium acetate trihydrate, 0.1 M sodium cacodylate trihydrate pH 6.5 and 30 % w/v PEG 8000). The well of the crystal tray was filled with 300 μL of the same solution. Fe(II)-Fe_BMb crystals were soaked in a cryoprotectant solution and flash frozen in liquid N₂ in a glove box before data collection.

Data collection

Diffraction data of Fe(II)-Fe_BMb were collected using Fe-edge absorption (1.7309 Å) as wavelengths of data collection at the Brookhaven National Synchrotron Light Source X12C beamline. All data were integrated using the program HKL2000³⁶.

Crystal structure determination

The crystal structure was solved by the molecular replacement method using MOLREP in the CCP4 Package³⁷. Refinement was performed using X-plor³⁸ and SHELX’97³⁹. The position of Fe²⁺ in the Fe_B site, and the E68 and H43 residues were rebuilt by the program O⁴⁰.

NO activity studies by GC/MS

NO reduction was monitored by GC/MS using an HP-Plot Q column (Aligent), installed in a Hewlett Packard 5890 gas chromatography system equipped with an online mass spectrometer (Micromass 70-VSE). As dithionite can reduce NO non-enzymatically, the NO activity of Fe_BMb was probed under one turnover conditions whereby the two electrons required for reduction, one from the reduced Fe(II) heme and one from the ferrous Fe_B site of Fe(II)-Fe_BMb, were rate limiting. Where applicable, an extra 2 molar equivalents of dithionite was added to the protein/metal solution to induce an additional turnover. Glucose, glucose oxidase and catalase were added to scavenge residual O₂ as described by Giuffre⁴¹. Under anaerobic conditions, a 2.67 mL reaction sample consisting of 0.6 mM protein, 10 mM glucose, glucose oxidase (100 μg/mL) and catalase (50 μg/mL) in 20 mM Bis Tris (treated with chelating agent) pH 7 was prepared in a Schlenk flask fitted with a septum. The head-space of the flask was then exchanged to helium. Purified NO gas (1 atm) was injected into the head space of the reaction flask via a gas-tight syringe and the reaction mixture was stirred at room temperature (the molar ratio of NO:protein = ~ 17:1). Typically, 250 μL of head space gas was analyzed via GC/MS at different time intervals to observe NO and N₂O levels. MS of NO (30 MW) and N₂O (44 MW) are shown in Fig. S6. NO₂ (46 MW) formation was not observed.