NO reduction by nitric-oxide reductase from denitrifying bacterium Pseudomonas aeruginosa: characterization of reaction intermediates that appear in the single turnover cycle

Nitric-oxide reductase (NOR) of a denitrifying bacterium catalyzes NO reduction to N(2)O at the binuclear catalytic center consisting of high spin heme b(3) and non-heme Fe(B). The structures of the reaction intermediates in the single turnover of the NO reduction by NOR from Pseudomonas aeruginosa were investigated using optical absorption and EPR spectroscopies combined with an originally designed freeze-quench device. In the EPR spectrum of the sample, in which the fully reduced NOR was mixed with an NO solution and quenched at 0.5 ms after the mixing, two characteristic signals for the ferrous Fe(B)-NO and the penta-coordinated ferrous heme b(3)-NO species were observed. The CO inhibition of its formation indicated that two NO molecules were simultaneously distributed into the two irons of the same binuclear center of the enzyme in this state. The time- and temperature-dependent EPR spectral changes indicated that the species that appeared at 0.5 ms is a transient reaction intermediate prior to the N(2)O formation, in good agreement with the so-called "trans" mechanism. It was also found that the final state of the enzyme in the single turnover cycle is the fully oxidized state, in which the mu-oxo-bridged ligand is absent between the two irons of its binuclear center, unlike the resting form of NOR as isolated. On the basis of these present findings, we propose a newly developed mechanism for the NO reduction reaction conducted by NOR.     

Structures of reduced and ligand‐bound nitric oxide reductase provide insights into functional differences in respiratory enzymes

Nitric oxide reductase (NOR) catalyzes the generation of nitrous oxide (N₂O) via the reductive coupling of two nitric oxide (NO) molecules at a heme/non‐heme Fe center. We report herein on the structures of the reduced and ligand‐bound forms of cytochrome c‐dependent NOR (cNOR) from Pseudomonas aeruginosa at a resolution of 2.3–2.7 Å, to elucidate structure‐function relationships in NOR, and compare them to those of cytochrome c oxidase (CCO) that is evolutionarily related to NOR. Comprehensive crystallographic refinement of the CO‐bound form of cNOR suggested that a total of four atoms can be accommodated at the binuclear center. Consistent with this, binding of bulky acetaldoxime (CH₃‐CH=N‐OH) to the binuclear center of cNOR was confirmed by the structural analysis. Active site reduction and ligand binding in cNOR induced only ～0.5 Å increase in the heme/non‐heme Fe distance, but no significant structural change in the protein. The highly localized structural change is consistent with the lack of proton‐pumping activity in cNOR, because redox‐coupled conformational changes are thought to be crucial for proton pumping in CCO. It also permits the rapid decomposition of cytotoxic NO in denitrification. In addition, the shorter heme/non‐heme Fe distance even in the bulky ligand‐bound form of cNOR (～4.5 Å) than the heme/Cu distance in CCO (～5 Å) suggests the ability of NOR to maintain two NO molecules within a short distance in the confined space of the active site, thereby facilitating N‐N coupling to produce a hyponitrite intermediate for the generation of N₂O. Proteins 2014; 82:1258–1271. © 2013 Wiley Periodicals, Inc.     

From NO to OO: Nitric Oxide and Dioxygen in Bacterial Respiration

Nitric oxide reductase (NOR) is a key enzyme in denitrification, reforming the N–N bond (making N₂O from two NO molecules) in the nitrogen cycle. It is a cytochrome bc complex which has apparently only two subunits, NorB and NorC. It contains two low-spin cytochromes (c and b), and a high-spin cytochrome b which forms a binuclear center with a non-heme iron. NorC contains the c-type heme and NorB can be predicted to bind the other metal centers. NorB is homologous to the major subunit of the heme/copper cytochrome oxidases, and NOR thus belongs to the superfamily, although it has an Fe/Fe active site rather than an Fe/Cu binuclear center and a different catalytic activity. Current evidence suggests that NOR is not a proton pump, and that the protons consumed in NO reduction are not taken from the cytoplasmic side of the membrane. Therefore, the comparison between structural and functional properties of NOR and cytochrome c- and quinol-oxidizing enzymes which function as proton pumps may help us to understand the mechanism of the latter. This review is a brief summary of the current knowledge on molecular biology, structure, and bioenergetics of NOR as a member of the oxidase superfamily.     

Reduction of nitric oxide in bacterial nitric oxide reductase--a theoretical model study

The mechanism of the nitric oxide reduction in a bacterial nitric oxide reductase (NOR) has been investigated in two model systems of the heme-b(3)-Fe(B) active site using density functional theory (B3LYP). A model with an octahedral coordination of the non-heme Fe(B) consisting of three histidines, one glutamate and one water molecule gave an energetically feasible reaction mechanism. A tetrahedral coordination of the non-heme iron, corresponding to the one of Cu(B) in cytochrome oxidase, gave several very high barriers which makes this type of coordination unlikely. The first nitric oxide coordinates to heme b(3) and is partly reduced to a more nitroxyl anion character, which activates it toward an attack from the second NO. The product in this reaction step is a hyponitrite dianion coordinating in between the two irons. Cleaving an NO bond in this intermediate forms an Fe(B) (IV)O and nitrous oxide, and this is the rate determining step in the reaction mechanism. In the model with an octahedral coordination of Fe(B) the intrinsic barrier of this step is 16.3 kcal/mol, which is in good agreement with the experimental value of 15.9 kcal/mol. However, the total barrier is 21.3 kcal/mol, mainly due to the endergonic reduction of heme b(3) taken from experimental reduction potentials. After nitrous oxide has left the active site the ferrylic Fe(B) will form a mu-oxo bridge to heme b(3) in a reaction step exergonic by 45.3 kcal/mol. The formation of a quite stable mu-oxo bridge between heme b(3) and Fe(B) is in agreement with this intermediate being the experimentally observed resting state in oxidized NOR. The formation of a ferrylic non-heme Fe(B) in the proposed reaction mechanism could be one reason for having an iron as the non-heme metal ion in NOR instead of a Cu as in cytochrome oxidase.     

Low-spin heme b(3) in the catalytic center of nitric oxide reductase from Pseudomonas nautica

Respiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mo?ssbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe(B)). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while Fe(B) was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mo?ssbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g ～ 6 and g ～ 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g ～ 6 region were assigned to a small quantity of uncoupled high-spin Fe(III) heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin-spin coupled binuclear center comprising the low-spin Fe(III) heme b(3) and the high-spin Fe(B)(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe(II)-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe(B)(II) site. The function of heme b(3) would then be to orient the Fe(B)-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction.     

Electrochemical assay for determining nitrosyl derivatives of human hemoglobin: nitrosylhemoglobin and S-nitrosylhemoglobin

Nitric oxide (NO) is an important biological regulator. It can bind to heme iron and form NO+, involved in the synthesis of S-nitrosothiols (-SNOs). NO reacts with human hemoglobin (Hb) to produce the derivatives: S-nitrosylhemoglobin (-SNOHb) and nitrosylhemoglobin (HbNO). At neutral pH values, free NO does not react directly with the -SH groups of Hb. The reductive nitrosylation of Fe(III) heme upon reaction with NO has long been studied, but it is not yet completely known. To quantify the reaction of NO with Hb, we developed a new, sensitive (nanomolar concentration range) electrochemical assay to selectively measure HbNO and -SNOHb. The assay also allows the monitoring of free NO during the reaction with human Fe(III)Hb and Fe(II)HbO(2).     

A low-redox potential heme in the dinuclear center of bacterial nitric oxide reductase: implications for the evolution of energy-conserving heme-copper oxidases.

Bacterial nitric oxide reductase (NOR) catalyzes the two-electron reduction of nitric oxide to nitrous oxide. It is a highly diverged member of the superfamily of heme-copper oxidases. The main feature by which NOR is distinguished from the heme-copper oxidases is the elemental composition of the active site, a dinuclear center comprised of heme b(3) and non-heme iron (Fe(B)). The visible region electronic absorption spectrum of reduced NOR exhibits a maximum at 551 nm with a distinct shoulder at 560 nm; these are attributed to Fe(II) heme c (E(m) = 310 mV) and Fe(II) heme b (E(m) = 345 mV), respectively. The electronic absorption spectrum of oxidized NOR exhibits a characteristic shoulder around 595 nm that exhibits complex behavior in equilibrium redox titrations. The first phase of reduction is characterized by an apparent shift of the shoulder to 604 nm and a decrease in intensity. This is due to reduction of Fe(B) (E(m) = 320 mV), while the subsequent bleaching of the 604 nm band represents reduction of heme b(3) (E(m) = 60 mV). This separation of redox potentials (>200 mV) allows the enzyme to be poised in the three-electron reduced state for detailed spectroscopic examination of the Fe(III) heme b(3) center. The low midpoint potential of heme b(3) represents a thermodynamic barrier to the complete (two-electron) reduction of the dinuclear center. This may avoid formation of a stable Fe(II) heme b(3)-NO species during turnover, which may be an inhibited state of the enzyme. It would also appear that the evolution of significant oxygen reducing activity by heme-copper oxidases was not simply a matter of the substitution of copper for non-heme iron in the dinuclear center. Changes in the protein environment that modulate the midpoint redox potential of heme b(3) to facilitate both complete reduction of the dinuclear center (a prerequisite for oxygen binding) and rapid heme-heme electron transfer were also necessary.     

Spectrophotometric activity microassay for pure and recombinant cytochrome P450-type nitric oxide reductase

Nitric oxide reductase (NOR) of the P450 oxidoreductase family accepts electrons directly from its cofactor, NADH, to reduce two nitric oxide (NO) molecules to one nitrous oxide molecule and water. The enzyme plays a key role in the removal of radical NO produced during respiratory metabolism, and applications in bioremediation and biocatalysis have been identified. However, a rapid, accurate, and sensitive enzyme assay has not yet been developed for this enzyme family. In this study, we optimized reaction conditions for the development of a spectrophotometric NOR activity microassay using NOC-5 for the provision of NO in solution. We also demonstrate that the assay is suitable for the quantification and characterization of P450-type NOR. The K_m and k_cat kinetic constants obtained by this assay were comparable to the values determined by gas chromatography, but with improved convenience and cost efficiency, effectively by miniaturization. To our knowledge, this is the first study to present the quantification of NOR activity in a kinetic microassay format.     

Proton and electron pathways in the bacterial nitric oxide reductase

Electron- and proton-transfer reactions in bacterial nitric oxide reductase (NOR) have been investigated by optical spectroscopy and electrometry. In liposomes, NOR does not show any generation of an electric potential during steady-state turnover. This electroneutrality implies that protons are taken up from the same side of the membrane as electrons during catalysis. Intramolecular electron redistribution after photolysis of the partially reduced CO-bound enzyme shows that the electron transfer in NOR has the same pathway as in the heme-copper oxidases. The electron is transferred from the acceptor site, heme c, via a low-spin heme b to the binuclear active site (heme b3/FeB). The electron-transfer rate between hemes c and b is (3 +/- 2) x 10(4) s(-1). The rate of electron transfer between hemes b and b3 is too fast to be resolved (>10(6) s(-1)). Only electron transfer between heme c and heme b is coupled to the generation of an electric potential. This implies that the topology of redox centers in NOR is comparable to that in the heme-copper cytochrome oxidases. The optical and electrometric measurements allow identification of the intermediate states formed during turnover of the fully reduced enzyme, as well as the associated proton and electron movement linked to the NO reduction. The first phase (k = 5 x 10(5) s(-1)) is electrically silent, and characterized by the disappearance of absorbance at 433 nm and the appearance of a broad peak at 410 nm. We assign this phase to the formation of a ferrous NO adduct of heme b3. NO binding is followed by a charge separation phase (k = 2.2 x 10(5) s(-1)). We suggest that the formation of this intermediate that is not linked to significant optical changes involves movement of charged side chains near the active site. The next step creates a negative potential with a rate constant of approximately 3 x 10(4) s(-1) and a weak optical signature. This is followed by an electrically silent phase with a rate constant of 5 x 10(3) s(-1) leading to the last intermediate of the first turnover (a rate constant of approximately 10(3) s(-1)). The fully reduced enzyme has four electrons, enough for two complete catalytic cycles. However, the protons for the second turnover must be taken from the bulk, resulting in the generation of a positive potential in two steps. The optical measurements also verify two phases in the oxidation of low-spin hemes. Based on these results, we present mechanistic models of NO reduction by NOR. The results can be explained with a trans mechanism rather than a cis model involving FeB. Additionally, the data open up the possibility that NOR employs a P450-type mechanism in which only heme b3 functions as the NO binding site during turnover.     

Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen

The respiratory nitric oxide reductase (NOR) from Paracoccus denitrificans catalyzes the two-electron reduction of NO to N(2)O (2NO + 2H(+) + 2e(-) --> N(2)O + H(2)O), which is an obligatory step in the sequential reduction of nitrate to dinitrogen known as denitrification. NOR has four redox-active cofactors, namely, two low-spin hemes c and b, one high-spin heme b(3), and a non-heme iron Fe(B), and belongs to same superfamily as the oxygen-reducing heme-copper oxidases. NOR can also use oxygen as an electron acceptor; this catalytic activity was investigated in this study. We show that the product in the steady-state reduction of oxygen is water. A single turnover of the fully reduced NOR with oxygen was initiated using the flow-flash technique, and the progress of the reaction monitored by time-resolved optical absorption spectroscopy. Two major phases with time constants of 40 micros and 25 ms (pH 7.5, 1 mM O(2)) were observed. The rate constant for the faster process was dependent on the O(2) concentration and is assigned to O(2) binding to heme b(3) at a bimolecular rate constant of 2 x 10(7) M(-)(1) s(-)(1). The second phase (tau = 25 ms) involves oxidation of the low-spin hemes b and c, and is coupled to the uptake of protons from the bulk solution. The rate constant for this phase shows a pH dependence consistent with rate limitation by proton transfer from an internal group with a pK(a) = 6.6. This group is presumably an amino acid residue that is crucial for proton transfer to the catalytic site also during NO reduction.     

Reduction of nitric oxide in bacterial nitric oxide reductase—a theoretical model study

The mechanism of the nitric oxide reduction in a bacterial nitric oxide reductase (NOR) has been investigated in two model systems of the heme-b₃-Fe_B active site using density functional theory (B3LYP). A model with an octahedral coordination of the non-heme Fe_B consisting of three histidines, one glutamate and one water molecule gave an energetically feasible reaction mechanism. A tetrahedral coordination of the non-heme iron, corresponding to the one of Cu_B in cytochrome oxidase, gave several very high barriers which makes this type of coordination unlikely. The first nitric oxide coordinates to heme b₃ and is partly reduced to a more nitroxyl anion character, which activates it toward an attack from the second NO. The product in this reaction step is a hyponitrite dianion coordinating in between the two irons. Cleaving an NO bond in this intermediate forms an Fe_B (IV)O and nitrous oxide, and this is the rate determining step in the reaction mechanism. In the model with an octahedral coordination of Fe_B the intrinsic barrier of this step is 16.3 kcal/mol, which is in good agreement with the experimental value of 15.9 kcal/mol. However, the total barrier is 21.3 kcal/mol, mainly due to the endergonic reduction of heme b₃ taken from experimental reduction potentials. After nitrous oxide has left the active site the ferrylic Fe_B will form a μ-oxo bridge to heme b₃ in a reaction step exergonic by 45.3 kcal/mol. The formation of a quite stable μ-oxo bridge between heme b₃ and Fe_B is in agreement with this intermediate being the experimentally observed resting state in oxidized NOR. The formation of a ferrylic non-heme Fe_B in the proposed reaction mechanism could be one reason for having an iron as the non-heme metal ion in NOR instead of a Cu as in cytochrome oxidase.     

Bacterial denitrifying nitric oxide reductases and aerobic respiratory terminal oxidases use similar delivery pathways for their molecular substrates

The superfamily of heme?copper oxidoreductases (HCOs) include both NO and O₂ reductases. Nitric oxide reductases (NORs) are bacterial membrane enzymes that catalyze an intermediate step of denitrification by reducing nitric oxide (NO) to nitrous oxide (N₂O). They are structurally similar to heme?copper oxygen reductases (HCOs), which reduce O₂ to water. The experimentally observed apparent bimolecular rate constant of NO delivery to the deeply buried catalytic site of NORs was previously reported to approach the diffusion-controlled limit (10⁸–10⁹?M^?1?s^?1). Using the crystal structure of cytochrome-c dependent NOR (cNOR) from Pseudomonas aeruginosa, we employed several protocols of molecular dynamics (MD) simulation, which include flooding simulations of NO molecules, implicit ligand sampling and umbrella sampling simulations, to elucidate how NO in solution accesses the catalytic site of this cNOR. The results show that NO partitions into the membrane, enters the enzyme from the lipid bilayer and diffuses to the catalytic site via a hydrophobic tunnel that is resolved in the crystal structures. This is similar to what has been found for O₂ diffusion through the closely related O₂ reductases. The apparent second order rate constant approximated using the simulation data is ~5?×?10⁸?M^?1?s^?1, which is optimized by the dynamics of the amino acid side chains lining in the tunnel. It is concluded that both NO and O₂ reductases utilize well defined hydrophobic tunnels to assure that substrate diffusion to the buried catalytic sites is not rate limiting under physiological conditions.     

Molecular structure and function of bacterial nitric oxide reductase

The crystal structure of the membrane-integrated nitric oxide reductase cNOR from Pseudomonas aeruginosa was determined. The smaller NorC subunit of cNOR is comprised of 1 trans-membrane helix and a hydrophilic domain, where the heme c is located, while the larger NorB subunit consists of 12 trans-membrane helices, which contain heme b and the catalytically active binuclear center (heme b(3) and non-heme Fe(B)). The roles of the 5 well-conserved glutamates in NOR are discussed, based on the recently solved structure. Glu211 and Glu280 appear to play an important role in the catalytic reduction of NO at the binuclear center by functioning as a terminal proton donor, while Glu215 probably contributes to the electro-negative environment of the catalytic center. Glu135, a ligand for Ca(2+) sandwiched between two heme propionates from heme b and b(3), and the nearby Glu138 appears to function as a structural factor in maintaining a protein conformation that is suitable for electron-coupled proton transfer from the periplasmic region to the active site. On the basis of these observations, the possible molecular mechanism for the reduction of NO by cNOR is discussed. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Structural insights into the molecular mechanism of H‐NOX activation

Nitric oxide (NO) signaling in mammals controls important processes such as smooth muscle relaxation and neurotransmission by the activation of soluble guanylate cyclase (sGC). NO binding to the heme domain of sGC leads to dissociation of the iron–histidine (Fe–His) bond, which is required for enzyme activity. The heme domain of sGC belongs to a larger class of proteins called H‐NOX (Heme‐Nitric oxide/OXygen) binding domains. Previous crystallographic studies on H‐NOX domains demonstrate a correlation between heme bending and protein conformation. It was unclear, however, whether these structural changes were important for signal transduction. Subsequent NMR solution structures of H‐NOX proteins show a conformational change upon disconnection of the heme and proximal helix, similar to those observed in the crystallographic studies. The atomic details of these conformational changes, however, are lacking in the NMR structures especially at the heme pocket. Here, a high‐resolution crystal structure of an H‐NOX mutant mimicking a broken Fe–His bond is reported. This mutant exhibits specific changes in heme conformation and major N‐terminal displacements relative to the wild‐type H‐NOX protein. Fe–His ligation is ubiquitous in all H‐NOX domains, and therefore, the heme and protein conformational changes observed in this study are likely to occur throughout the H‐NOX family when NO binding leads to rupture of the Fe–His bond.     

Reaction of carbon monoxide with the reduced active site of bacterial nitric oxide reductase.

Bacterial nitric oxide reductase (NOR), a member of the superfamily of heme-copper oxidases, catalyzes the two-electron reduction of nitric oxide to nitrous oxide. The key feature that distinguishes NOR from the typical heme-copper oxidases is the elemental composition of the dinuclear center, which contains non-heme iron (FeB) rather than copper (CuB). UV-vis electronic absorption and room-temperature magnetic circular dichroism (RT-MCD) spectroscopies showed that CO binds to Fe(II) heme b3 to yield a low-spin six-coordinate species. Photolysis of the Fe(II)-CO bond is followed by CO recombination (k(on) = 1.7 x 10(8) M(-1) x s(-1)) that is approximately 3 orders of magnitude faster than CO recombination to the active site of typical heme-copper oxidases (k(on) = 7 x 10(4) M(-1)x s(-1)). This rapid rate of CO recombination suggests an unimpeded pathway to the active site that may account for the enzyme's high affinity for substrate, essential for maintaining denitrification at low concentrations of NO. In contrast, the initial binding of CO to reduced heme b3 measured by stopped-flow spectroscopy is much slower (k(on) = 1.2 x 10(5) M(-1) x s(-1)). This suggests that an existing heme distal ligand (water/OH-) may be displaced to elicit the spin-state change observed in the RT-MCD spectrum.     

Substrate Control of Internal Electron Transfer in Bacterial Nitric-oxide Reductase

Nitric -oxide reductase (NOR) from Paracoccus denitrificans catalyzes the reduction of nitric oxide (NO) to nitrous oxide (N₂O) (2NO + 2H⁺ + 2e⁻ →N₂O + H₂O) by a poorly understood mechanism. NOR contains two low spin hemes c and b, one high spin heme b₃, and a non-heme iron Fe_B. Here, we have studied the reaction between fully reduced NOR and NO using the “flow-flash” technique. Fully (four-electron) reduced NOR is capable of two turnovers with NO. Initial binding of NO to reduced heme b₃ occurs with a time constant of ∼1 μs at 1.5 mm NO, in agreement with earlier studies. This reaction is [NO]-dependent, ruling out an obligatory binding of NO to Fe_B before ligation to heme b₃. Oxidation of hemes b and c occurs in a biphasic reaction with rate constants of 50 s⁻¹ and 3 s⁻¹ at 1.5 mm NO and pH 7.5. Interestingly, this oxidation is accelerated as [NO] is lowered; the rate constants are 120 s⁻¹ and 12 s⁻¹ at 75 μm NO. Protons are taken up from solution concomitantly with oxidation of the low spin hemes, leading to an acceleration at low pH. This effect is, however, counteracted by a larger degree of substrate inhibition at low pH. Our data thus show that substrate inhibition in NOR, previously observed during multiple turnovers, already occurs during a single oxidative cycle. Thus, NO must bind to its inhibitory site before electrons redistribute to the active site. The further implications of our data for the mechanism of NO reduction by NOR are discussed.     

Ambidentate H-bonding by heme-bound NO: structural and spectral effects of –O versus –N H-bonding

Resonance Raman studies have uncovered puzzling complexities in the structures of NO adducts of heme proteins. Although CO adducts of heme proteins obey well-behaved anti-correlations between Fe–C and C–O stretching frequencies, which reflect changes in backbonding induced by distal H-bonding residues, the corresponding NO data are scattered. This scatter can be traced to distal influences, since protein-free NO–hemes do show well-behaved anti-correlations. Why do distal effects produce irregularities in νFeN/νNO plots but not in νFeC/νCO plots? We show via density functional theory (DFT) computations on model systems that the response to distal H-bonding differs markedly when the NO acceptor atom is N versus O. Backbonding is augmented by H-bonding to O, but the effect of H-bonding to N is to weaken both N–O and N–Fe bonds. The resulting downward deviation from the νFeN/νNO backbonding line increases with increasing H-bond strength. This effect explains the deviations observed for a series of myoglobin variants, in which the strength of distal H-bonding is modulated by distal pocket residue substitutions. Most of the data follow a positive νFeN/νNO correlation with the same slope as that calculated for H-bonding to N. Such deviations are not observed for CO adducts, because the CO π* orbital is unoccupied, and serves as a delocalized acceptor of H-bonds. H-bonding to N primes NO–heme for reduction to the HNO adduct, a putative intermediate in NO-reducing enzymes.