The bacterial manganese oxidase MnxG of the Mnx protein complex is unique among multicopper oxidases (MCOs) in carrying out a two-electron metal oxidation, converting Mn(II) to MnO2 nanoparticles. The reaction occurs in two stages: Mn(II) → Mn(III) and Mn(III) → MnO2. In a companion study, we show that the electron transfer from Mn(II) to the low-potential type 1 Cu of MnxG requires an activation step, likely forming a hydroxide bridge at a dinuclear Mn(II) site. Here we study the second oxidation step, using pyrophosphate (PP) as a Mn(III) trap. PP chelates Mn(III) produced by the enzyme and subsequently allows it to become a substrate for the second stage of the reaction. EPR spectroscopy confirms the presence of Mn(III) bound to the enzyme. The Mn(III) oxidation step does not involve direct electron transfer to the enzyme from Mn(III), which is shown by kinetic measurements to be excluded from the Mn(II) binding site. Instead, Mn(III) is proposed to disproportionate at an adjacent polynuclear site, thereby allowing indirect oxidation to Mn(IV) and recycling of Mn(II). PP plays a multifaceted role, slowing the reaction by complexing both Mn(II) and Mn(III) in solution, and also inhibiting catalysis, likely through binding at or near the active site. An overall mechanism for Mnx-catalyzed MnO2 production from Mn(II) is presented.

The bacterial protein complex Mnx contains a multicopper oxidase (MCO) MnxG that, unusually, catalyzes the two-electron oxidation of Mn(II) to MnO2 biomineral, via a Mn(III) intermediate. Although Mn(III)/Mn(II) and Mn(IV)/Mn(III) reduction potentials are expected to be high, we find a low reduction potential, 0.38 V (vs Normal Hydrogen Electrode, pH 7.8), for the MnxG type 1 Cu2+, the electron acceptor. Indeed the type 1 Cu2+ is not reduced by Mn(II) in the absence of molecular oxygen, indicating that substrate oxidation requires an activation step. We have investigated the enzyme mechanism via electronic absorption spectroscopy, using chemometric analysis to separate enzyme-catalyzed MnO2 formation from MnO2 nanoparticle aging. The nanoparticle aging time course is characteristic of nucleation and particle growth; rates for these processes followed expected dependencies on Mn(II) concentration and temperature, but exhibited different pH optima. The enzymatic time course is sigmoidal, signaling an activation step, prior to turnover. The Mn(II) concentration and pH dependence of a preceding lag phase indicates weak Mn(II) binding. The activation step is enabled by a pKa > 8.6 deprotonation, which is assigned to Mn(II)-bound H2O; it induces a conformation change (consistent with a high activation energy, 106 kJ/mol) that increases Mn(II) affinity. Mnx activation is proposed to decrease the Mn(III/II) reduction potential below that of type 1 Cu(II/I) by formation of a hydroxide-bridged binuclear complex, Mn(II)(μ-OH)Mn(II), at the substrate site. Turnover is found to depend cooperatively on two Mn(II) and is enabled by a pKa 7.6 double deprotonation. It is proposed that turnover produces a Mn(III)(μ-OH)2Mn(III) intermediate that proceeds to the enzyme product, likely Mn(IV)(μ-O)2Mn(IV) or an oligomer, which subsequently nucleates MnO2 nanoparticles. We conclude that Mnx exploits manganese polynuclear chemistry in order to facilitate an otherwise difficult oxidation reaction, as well as biomineralization. The mechanism of the Mn(III/IV) conversion step is elucidated in an accompanying paper.

The use of hybrid hemoglobin (Hb), with mesoheme substituted for protoheme, allows separate monitoring of the α or β hemes along the allosteric pathway. Using resonance Raman (rR) spectroscopy in silica gel, which greatly slows protein motions, we have observed that the Fe–histidine stretching frequency, νFeHis, which is a monitor of heme reactivity, evolves between frequencies characteristic of the R and T states, for both α or β chains, prior to the quaternary R–T and T–R shifts. Computation of νFeHis, using QM/MM and the conformational search program PELE, produced remarkable agreement with experiment. Analysis of the PELE structures showed that the νFeHis shifts resulted from heme distortion and, in the α chain, Fe–His bond tilting. These results support the tertiary two-state model of ligand binding (Henry et al., Biophys. Chem. 2002, 98, 149). Experimentally, the νFeHis evolution is faster for β than for α chains, and pump–probe rR spectroscopy in solution reveals an inflection in the νFeHis time course at 3 μs for β but not for α hemes, an interval previously shown to be the first step in the R–T transition. In the α chain νFeHis dropped sharply at 20 μs, the final step in the R–T transition. The time courses are fully consistent with recent computational mapping of the R–T transition via conjugate peak refinement by Karplus and co-workers (Fischer et al., Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 5608). The effector molecule IHP was found to lower νFeHis selectively for α chains within the R state, and a binding site in the α1α2 cleft is suggested.

The recently developed technique of femtosecond stimulated Raman spectroscopy, and its variant, femtosecond Raman-induced Kerr effect spectroscopy (FRIKES), offer access to ultrafast excited-state dynamics via structurally specific vibrational spectra. We have used FRIKES to study the photoexcitation dynamics of nickel(II) phthalocyanine with eight butoxy substituents, NiPc(OBu)8. NiPc(OBu)8 is reported to have a relatively long-lived ligand-to-metal charge-transfer (LMCT) state, an essential characteristic for efficient electron transfer in photocatalysis. Following photoexcitation, vibrational transitions in the FRIKES spectra, assignable to phthalocyanine ring modes, evolve on the femtosecond to picosecond time scales. Correlation of ring core size with the frequency of the ν10 (asymmetric C–N stretching) mode confirms the identity of the LMCT state, which has a ∼500 ps lifetime, as well as that of a precursor d-d excited state. An even earlier (∼0.2 ps) transient is observed and tentatively assigned to a higher-lying Jahn–Teller-active LMCT state. This study illustrates the power of FRIKES spectroscopy in elucidating ultrafast molecular dynamics.

The gaseous XO molecules (X = C, N or O) bind to the heme prosthetic group of heme proteins, and thereby activate or inhibit key biological processes. These events depend on interactions of the surrounding protein with the FeXO adduct, interactions that can be monitored via the frequencies of the FeX and XO bond stretching modes, νFeX and νXO. The frequencies can be determined by vibrational spectroscopy, especially resonance Raman spectroscopy. Backbonding, the donation of Fe dπ electrons to the XO π* orbitals, is a major bonding feature in all the FeXO adducts. Variations in backbonding produce negative νFeX/νXO correlations, which can be used to gauge electrostatic and H-bonding effects in the protein binding pocket. Backbonding correlations have been established for all the FeXO adducts, using porphyrins with electron donating and withdrawing substituents. However the adducts differ in their response to variations in the nature of the axial ligand, and to specific distal interactions. These variations provide differing vantages for evaluating the nature of protein–heme interactions. We review experimental studies that explore these variations, and DFT computational studies that illuminate the underlying physical mechanisms.Highlights► Negative correlations between FeX and XO stretching frequencies establish backbonding in XO adducts (X = C, N, O) of heme proteins. ► For Fe(II)CO adducts, donation from the trans axial ligand decreases νCO, with little change in νFeC, and shifts the backbonding correlation. ► For Fe(III)NO, Fe(II)NO and Fe(II)O2, dz2–dxzdz2–dxz orbital mixing produces positive νFeX/νXO correlations, as the trans donation increases. ► For Fe(II)NO and Fe(II)O2, negative or positive correlations are induced by H-bonding to the outer or inner XO atom. ► Fe(III)NO adducts do not support distal H-bonds, but do support lone pair donor interactions. Strong donors interact with N, bending the FeNO.

The gaseous ligands, CO, NO, and O2 interact with the Fe ion in heme proteins largely via backbonding of Fe electrons to the π* orbitals of the XO (X = C, N, O) ligands. In these FeXO adducts, the Fe–X stretching frequency varies inversely with the X–O stretching frequency, since increased backbonding strengthens the Fe–X bond while weakening the X–O bond. Inverse frequency correlations have been observed for all three ligands, despite differing electronic and geometric structures, and despite variable composition of the “FeX” vibrational mode, in which Fe–X stretching and Fe–X–O coordinates are mixed for bent FeXO adducts. We report experimental data for 5-coordinate CoII(NO) porphyrin adducts (isoelectronic with FeII(OO) adducts), and the results of density functional theory (DFT) modeling for 5-coordinate FeII(NO), CoII(NO), and FeII(OO) adducts. Inverse ν(MX)/ν(XO) correlations are obtained computationally, using model porphyrins with graded electron-donating and -withdrawing substituents to modulate the backbonding. Computed slopes agree satisfactorily with experiment, provided nonhybrid functionals are used, which avoid overemphasizing high-spin states. The BP86 functional gives correct ground states, a closed-shell singlet for CoII(NO) and an open-shell singlet for the isoelectronic FeII(OO), as corroborated by structural data for CoII(NO), and the ν(MX)/ν(XO) slope agreement with experiment for both adducts. However, for FeII(OO) adducts, the computed inverse ν(MX)/ν(XO) correlation applies only to porphyrins with electron-donating and withdrawing substituents of moderate strength. For substituents more donating than −CH3, a direct correlation is obtained, the Fe–O and O–O bonds weakening in concert. This effect is ascribed to the dominance of σ bonding via the in-plane dxz(+dz2)-π* orbital, when electron-donating substituents raise the d orbital energies sufficiently to render backbonding (dyz-π*) unimportant.

Encapsulation of hemoglobin (Hb) in silica gel preserves structure and function but greatly slows protein motion, thereby providing access to intermediates along the allosteric pathway that are inaccessible in solution. Resonance Raman (RR) spectroscopy with visible and ultraviolet laser excitation provides probes of heme reactivity and of key tertiary and quaternary contacts. These probes were monitored in gels after deoxygenation of oxyHb and after CO binding to deoxyHb, which initiate conformational change in the R–T and T–R directions, respectively. The spectra establish that quaternary structure change in the gel takes a week or more but that the evolution of heme reactivity, as monitored by the Fe–histidine stretching vibration, νFeHis, is completed within two days, and is therefore uncoupled from the quaternary structure. Within each quaternary structure, the evolving νFeHis frequencies span the full range of values between those previously associated with the high- and low-affinity end states, R and T. This result supports the tertiary two-state (TTS) model, in which the Hb subunits can adopt high- and low-affinity tertiary structures, r and t, within each quaternary state. The spectra also reveal different tertiary pathways, involving the breaking and reformation of E and F interhelical contacts in the R–T direction but not the T–R direction. In the latter, tertiary motions are restricted by the T quaternary contacts.

Cytochrome c unfolds locally and reversibly upon heating at pH 3. UV resonance Raman (UVRR) spectra reveal that instead of producing unordered structure, unfolding converts turns and some helical elements to β-sheet. It also disrupts the Met80–heme bond, and has been previously shown to induce peroxidase activity. Aromatic residues that are H-bonded to a heme propionate (Trp59 and Tyr48) alter their orientation, indicating heme displacement. T-jump/UVRR measurements give time constants of 0.2, 3.9, and 67 μs for successive phases of β-sheet formation and concomitant reorientation of Trp59. UVRR spectra reveal protonation of histidines, and specifically of His26, whose H-bond to Pro44 anchors the 40s Ω loop; this loop is known to be the least stable ‘foldon’ in the protein. His26 protonation is proposed to disrupt its H-bond with Pro44, triggering the extension of a short β-sheet segment at the ‘neck’ of the 40s Ω loop into the loop itself and back into the 60s and 70s helices. The secondary structure change displaces the heme via H-bonds from residues in the growing β-sheet, thereby exposing it to exogenous ligands, and inducing peroxidase activity. This unfolding mechanism may play a role in cardiolipin peroxidation by cyt c during apoptosis.

The affinity and reactivity of the gaseous molecules CO, NO and O2 (XO) in heme protein adducts are controlled by secondary interactions, especially by H-bonds donated from distal protein residues. Vibrational spectroscopy, supported by DFT (Density Functional Theory) modeling, has revealed that for NO and O2, but not for CO, a critical issue is whether the H-bond is donated to the outer or inner atom of the bound diatomic ligand. DFT modeling shows that bound NO and O2 are ambidentate, both atoms separately acting as H-bond acceptors. This is not the case for CO, whose π* orbital acts as a delocalized H-bond acceptor. Vibrational spectra of heme-XO adducts reveal a general pattern of backbonding variations, marked by families of negative correlations between frequencies associated with FeX and XO bond stretches. For heme-CO adducts, H-bonding increases backbonding, the νFeX/νXO points moving up the backbonding correlation established with model compounds. For NO and O2 adducts, however, increased backbonding is only observed when the outer atom is the H-bond acceptor. H-bonding to the inner (X) atom instead produces a positive νFeX/νXO correlation. This effect can be reproduced by DFT modeling. Its mechanism is polarization of the sp2 orbital on the X atom, on the back side of the bent FeXO unit, drawing electrons from both the FeX and XO bonds and weakening them together. Thus, the positioning of H-bond donors in the protein differentially affects bonding and reactivity in heme adducts of NO and O2.The positioning of H-bond donors by the protein differentially controls bonding and reactivity in heme‐NO and -O2 adducts. This article reviews the results of vibrational spectroscopy and DFT modeling, revealing a critical issue of whether the H-bond is donated to the outer or inner atom of the bound ligand.Highlights► H-bonding in heme protein adducts of XO (X=C, N, O) are analyzed with DFT and RR. ► Different response of FeNO and FeO2 to the position of H-bond donors is revealed. ► H-bonding from heme pocket residues to the outer oxygen increases backbonding. ► H-bonding to the inner atom of FeXO weakens the Fe-X and X-O bonds in concert. ► Positioning of H-bond donors by the protein controls bonding in heme FeNO and FeO2.

Global cycling of environmental manganese requires catalysis by bacteria and fungi for MnO2 formation, since abiotic Mn(II) oxidation is slow under ambient conditions. Genetic evidence from several bacteria indicates that multicopper oxidases (MCOs) are required for MnO2 formation. However, MCOs catalyze one-electron oxidations, whereas the conversion of Mn(II) to MnO2 is a two-electron process. Trapping experiments with pyrophosphate (PP), a Mn(III) chelator, have demonstrated that Mn(III) is an intermediate in Mn(II) oxidation when mediated by exosporium from the Mn-oxidizing bacterium Bacillus SG-1. The reaction of Mn(II) depends on O2 and is inhibited by azide, consistent with MCO catalysis. We show that the subsequent conversion of Mn(III) to MnO2 also depends on O2 and is inhibited by azide. Thus, both oxidation steps appear to be MCO-mediated, likely by the same enzyme, which is indicated by genetic evidence to be the MnxG gene product. We propose a model of how the manganese oxidase active site may be organized to couple successive electron transfers to the formation of polynuclear Mn(IV) complexes as precursors to MnO2 formation.

The imidazole side-chains of histidine residues perform key roles in proteins, and spectroscopic markers are of great interest. The imidazole Raman spectrum is subject to resonance enhancement at UV wavelengths, and a number of UVRR markers of structure have been investigated. We report a systematic experimental and computational study of imidazole UVRR spectra, which elucidates the band pattern, and the effects of protonation and deprotonation, of H/D exchange, of metal complexation, and of addition of a methyl substituent, modeling histidine itself. A consistent assignment scheme is proposed, which permits tracking of the bands through these chemical variations. The intensities are dominated by normal mode contributions from stretching of the strongest ring bonds, C2N and C4C5, consistent with enhancement via resonance with a dominant imidazole π–π* transition.

Visible and ultraviolet resonance Raman (RR) spectra are reported for FeIII(NO) adducts of myoglobin variants with altered polarity in the distal heme pockets. The stretching frequencies of the FeIII−NO and N−O bonds, νFeN and νNO, are negatively correlated, consistent with backbonding. However, the correlation shifts to lower νNO for variants lacking a distal histidine. DFT modeling reproduces the shifted correlations and shows the shift to be associated with the loss of a lone-pair donor interaction from the distal histidine that selectively strengthens the N−O bond. However, when the model contains strongly electron-withdrawing substituents at the heme β-positions, νFeN and νNO become positively correlated. This effect results from FeIII−N−O bending, which is induced by lone-pair donation to the NNO atom. Other mechanisms for bending are discussed, which likewise lead to a positive νFeN/νNO correlation, including thiolate ligation in heme proteins and electron-donating meso-substituents in heme models. The νFeN/νNO data for the Fe(III) complexes are reporters of heme pocket polarity and the accessibility of lone pair, Lewis base donors. Implications for biologically important processes, including NO binding, reductive nitrosylation, and NO reduction, are discussed.

Soluble guanylate cyclase (sGC) is weakly activated by carbon monoxide (CO) but is significantly activated by the binding of YC-1 to the sGC−CO complex. In this report, resonance Raman (RR) spectroscopy was used to study selected sGC variants. Addition of YC-1 to the sGC−CO complex alters the intensity pattern of RR bands assigned to the vinyl and propionate heme substituents, suggesting changes in the tilting of the pyrrole rings to which they are attached. YC-1 also shifts the RR intensity of the νFeC and νCO bands from 473 and 1985 cm−1 to 487 and 1969 cm−1, respectively, and induces an additional νFeC band, at 521 cm−1, assigned to five-coordinate heme-CO. Site-directed variants in the proximal heme pocket (P118A) or in the distal heme pocket (V5Y and I149Y) reduce the extent of YC-1 activation, along with the 473 cm−1 band intensity. These lower-activity sGC variants display another νFeC band at 493 cm−1 which is insensitive to YC-1 addition and is attributed to protein that cannot be activated by the allosteric activator. The results are consistent with a model in which YC-1 binding to the sGC−CO complex results in a conformational change that activates the protein. Specifically, YC-1 binding alters the heme geometry via peripheral nonbonded contacts and also relieves an intrinsic electronic effect that weakens FeCO backbonding in the native, YC-1 responsive protein. This electronic effect might involve neutralization of the heme propionates via H-bond contacts or negative polarization by a distal cysteine residue. YC-1 binding also strains the Fe−histidine bond, leading to a population of the five-coordinate sGC−CO complex in addition to a conformationally distinct population of the six-coordinate sGC−CO complex. The loss of YC-1 activation in the sGC variants might involve a weakening of the heme−protein contacts that are thought to be critical to a YC-1-induced conformational change.

Modulation of soluble guanylate cyclase (sGC) activity by nitric oxide (NO) involves two distinct steps. Low-level activation of sGC is achieved by the stoichiometric binding of NO (1-NO) to the heme cofactor, while much higher activation is achieved by the binding of additional NO (xsNO) at a non-heme site. Addition of the allosteric activator YC-1 to the 1-NO form leads to activity comparable to that of the xsNO state. In this study, the mechanisms of sGC activation were investigated using electronic absorption and resonance Raman (RR) spectroscopic methods. RR spectroscopy confirmed that the 1-NO form contains five-coordinate NO-heme and showed that the addition of NO to the 1-NO form has no significant effect on the spectrum. In contrast, addition of YC-1 to either the 1-NO or xsNO forms alters the RR spectrum significantly, indicating a protein-induced change in the heme geometry. This change in the heme geometry was also observed when BAY 41-2272 was added to the xsNO form. Bands assigned to bending and stretching motions of the vinyl and propionate substituents undergo changes in intensity in a pattern suggesting altered tilting of the pyrrole rings to which they are attached. In addition, the N−O stretching frequency increases, with no change in the Fe−NO stretching frequency, an effect modeled via DFT calculations as resulting from a small opening of the Fe−N−O angle. These spectral differences demonstrate different mechanisms of activation by synthetic activators, such as YC-1 and BAY 41-2272, and excess NO.

Cystathionine β-synthase (CBS) plays a central role in homocysteine metabolism, and malfunction of the enzyme leads to homocystinuria, a devastating metabolic disease. CBS contains a pyridoxal 5′-phosphate (PLP) cofactor which catalyzes the synthesis of cystathionine from homocysteine and serine. Mammalian forms of the enzyme also contain a heme group, which is not involved in catalysis. It may, however, play a regulatory role, since the enzyme is inhibited when CO or NO are bound to the heme. We have investigated the mechanism of this inhibition using fluorescence and resonance Raman spectroscopies. CO binding is found to induce a tautomeric shift of the PLP from the ketoenamine to the enolimine form. The ketoenamine is key to PLP reactivity because its imine C═N bond is protonated, facilitating attack by the nucleophilic substrate, serine. The same tautomer shift is also induced by heat inactivation of Fe(II)CBS, or by an Arg266Met replacement in Fe(II)CBS, which likewise inactivates the enzyme; in both cases the endogenous Cys52 ligand to the heme is replaced by another, unidentified ligand. CO binding also displaces Cys52 from the heme. We propose that the tautomer shift results from loss of a stabilizing H-bond from Asn149 to the PLP ring O3′ atom, which is negatively charged in the ketoenamine tautomer. This loss would be induced by displacement of the PLP as a result of breaking the salt bridge between Cys52 and Arg266, which resides on a short helix that is also anchored to the PLP via H-bonds to its phosphate group. The salt bridge would be broken when Cys52 is displaced from the heme. Cys52 protonation is inferred to be the rate-limiting step in breaking the salt bridge, since the rate of the tautomer shift, following CO binding, increases with decreasing pH. In addition, elevation of the concentration of phosphate buffer was found to diminish the rate and extent of the tautomer shift, suggesting a ketoenamine-stabilizing phosphate binding site, possibly at the protonated imine bond of the PLP. Implications of these findings for CBS regulation are discussed.

Hemoglobin (Hb) is an allosteric tetrameric protein made up of αβ heterodimers. The α and β chains are similar, but are chemically and structurally distinct. To investigate dynamical differences between the chains, we have prepared tetramers in which the chains are isotopically distinguishable, via reconstitution with 15N-heme. Ligand recombination and heme structural evolution, following HbCO dissociation, was monitored with chain selectivity by resonance Raman (RR) spectroscopy. For α but not for β chains, the frequency of the ν4 porphyrin breathing mode increased on the microsecond time scale. This increase is a manifestation of proximal tension in the Hb T-state, and its time course is parallel to the formation of T contacts, as determined previously by UVRR spectroscopy. Despite the localization of proximal constraint in the α chains, geminate recombination was found to be equally probable in the two chains, with yields of 39 ± 2%. We discuss the possibility that this equivalence is coincidental, in the sense that it arises from the evolutionary pressure for cooperativity, or that it reflects mechanical coupling across the αβ interface, evidence for which has emerged from UVRR studies of site mutants.

Time-resolved resonance Raman (RR) spectra are reported for hemoglobin (Hb) tetramers, in which the α and β chains are selectively substituted with mesoheme. The Soret absorption band shift in mesoheme relative to protoheme permits chain-selective recording of heme RR spectra. The evolution of these spectra following HbCO photolysis shows that the geminate recombination rates and the yields are the same for the two chains, consistent with recent results on 15N-heme isotopomer hybrids. The spectra also reveal systematic shifts in the deoxyheme ν4 and νFe–His RR bands, which are anticorrelated. These shifts are resolved for the successive intermediates in the protein structure, which have previously been determined from time-resolved UV RR spectra. Both chains show Fe–His bond compression in the immediate photoproduct, which relaxes during the formation of the first intermediate, Rdeoxy (0.07 μs), in which the proximal F-helix is proposed to move away from the heme. Subsequently, the Fe–His bond weakens, more so for the α chains than for the β chains. The weakening is gradual for the β chains, but is abrupt for the α chains, coinciding with completion of the R–T quaternary transition, at 20 μs. Since the transition from fast- to slow-rebinding Hb also occurs at 20 μs, the drop in the α chain νFe–His supports the localization of ligation restraint to tension in the Fe–His bond, at least in the α chains. The mechanism is more complex in the β chains.

Proteins in the H-NOX family act as sensors of NO or O2. This family includes soluble guanylate cyclase (sGC), the NO sensor that is responsible for vasodilation and neurotransmission in mammals. The crystal structures of bacterial H-NOX domains have revealed a highly distorted heme cofactor. This distortion has now been shown to be associated with a concerted displacement of the entire N-terminal half of the protein. This displacement likely provides the mechanism for transducing the ligand binding event into signaling.

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.