Correlations among parameters that describe low-spin ferric heme complexes

The g values from low-spin ferric hemes can be related through the t2g hole model to rhombic (V/lambda) and tetragonal (delta/lambda) ligand field components and to the lowest Kramer's doublet energy (E/lambda). The latter is also a measure of unpaired electron sharing among the iron 3d (t2g) orbitals. For a series of ligands (X), there is a monotonic increase in myoglobin complex (Mb . X) [E/lambda] values with nonheme hexacoordinate metal complex (M . X6) [eg-t2gPg] orbital separations. As the aqueous solution pKa values of the sulfurous or nitrogenous ligands in model heme complexes increase, values of V/lambda and delta/lambda increase linearly, but those of [E/lambda] decrease linearly. The greater the electron-acceptor ability of the ligand, as suggested by its position in the spectrochemical series or its pKa, the more the unpaired electron sharing among the heme t2g orbitals increases. The rate of change of [E/lambda] with V/lambda and the pKa is different with sulfurous and nitrogenous ligands, and the magnitude of both rates increases with two sulfurs less than sulfur and nitrogen less than two nitrogens bound to the heme. The maximum magnitude of this rate with V/lambda for cytochrome P-450 is four times less than that for myoglobin, which may explain, in part, the differences in ligand binding between these two hemeproteins. The perturbation of [E/lambda], V/lambda, and delta/lambda induced by strain of iron-ligand bonds is quantitated for several hemeproteins and heme models. In addition, energy level comparisons suggest that the largest-magnitude g value falls approximately along the iron-chlorin ring normal. This suggestion implies that the electron distribution of the iron at the catalytic sites of cytochrome P-450 and certain chlorin-containing enzymes is in some way similar, but distinct from that at the transport site of myoglobin.     

The identification of histidine ligands to cytochrome a in cytochrome c oxidase

A histidine auxotroph of Saccharomyces cerevisiae has been used to metabolically incorporate [1,3-15N2] histidine into yeast cytochrome c oxidase. Electron nuclear double resonance (ENDOR) spectroscopy of cytochrome a in the [15N]histidine-substituted enzyme reveals an ENDOR signal which can be assigned to hyperfine coupling of a histidine 15N with the low-spin heme, thereby unambiguously identifying histidine as an axial ligand to this cytochrome. Comparison of this result with similar ENDOR data obtained on two 15N-substituted bisimidazole model compounds, metmyoglobin-[15N]imidazole and bis[15N]imidazole tetraphenyl porphyrin, provides strong evidence for bisimidazole coordination in cytochrome a.     

Spectral properties of a novel cytochrome P-450 of a Saccharomyces cerevisiae mutant SG1. A cytochrome P-450 species having a nitrogenous ligand trans to thiolate

An altered cytochrome P-450 (SG1 P-450) was partially purified from Saccharomyces cerevisiae mutant SG1 which is defective in lanosterol 14 alpha-demethylation. Oxidized SG1 P-450 showed a Soret peak at 422 nm and the alpha peak was lower than the beta peak. This spectrum was considerably different from those of known low-spin P-450s, indicating a unique ligand structure of SG1 P-450. The absorption spectrum of ferric SG1 P-450 was superimposable on that of the imidazole complex of ferric P-450, suggesting the presence of a nitrogenous ligand such as histidine of the apoprotein at the 6th coordination position. SG1 P-450 was immunochemically indistinguishable from cytochrome P-450 of S. cerevisiae catalyzing lanosterol 14 alpha-demethylation (P-45014DM) but had no lanosterol 14 alpha-demethylase activity.     

Exogenous heme restores in vivo functional capacity of hepatic cytochrome P-450 destroyed by allylisopropylacetamide.

Administration of allylisopropylacetamide (AIA) produces a dose-related destruction of the heme moiety of the phenobarbital-induced subspecies of hepatic cytochrome P-450. This results in delayed plasma disappearance of the inactivating agent as determined after injection of [¹⁴C]AIA. In phenobarbital-pretreated rats, infusion of heme reversed this AIA-mediated impairment of the plasma disappearance of [¹⁴C]AIA. In the absence of phenobarbital pretreatment, cytochrome P-450 destruction by AIA was minimal and heme infusion failed to enhance plasma disappearance of [¹⁴C]AIA. Since exogenously administered heme is incorporated into hepatic cytochrome P-450 $\text{in vivo}$, these observations suggest that the infused heme restored the functional capacity of the phenobarbital-induced mixed function oxidase system by substituting for the prosthetic heme moiety destroyed by AIA. Heme infusion is a potentially useful therapeutic modality for enhancing drug biotransformation after intoxication with compounds that inactivate cytochrome P-450.     

Yeast cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation. I. Purification and spectral properties

A form of cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation (tentatively called "P-450(14)DM") was purified from microsomes of semi-anaerobically grown cells of Saccharomyces cerevisiae to gel electrophoretic homogeneity. An apparent monomeric Mr = 58,000 was estimated for the purified cytochrome by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both optical and EPR spectra of oxidized P-450(14)DM are characteristic of low spin ferric heme proteins, and its reduced CO complex showed a Soret absorption peak at 447 nm. As in the case of hepatic microsomal cytochromes P-450, the ethyl isocyanide complex of reduced P-450(14)DM was in a pH-dependent equilibrium between two states having Soret peaks at 429 and 453 nm, the equilibrium being considerably shifted toward the 453-nm state. Oxidized P-450(14)DM was peculiar in that in its CD spectrum there was a negative shoulder at 425 nm and the 350- and 414-nm troughs possessed larger and relatively smaller [theta] values, respectively, than those reported for other low spin ferric cytochromes P-450. Lanosterol was the only compound which caused a Type I spectral change in oxidized P-450(14)DM. The lanosterol-induced low to high spin state change was, however, only slight even at saturating concentrations of the sterol, indicating that the lanosterol-P-450(14)DM adduct was in a spin state equilibrium.     

1H nuclear magnetic resonance and magnetic circular dichroism studies of ferric low-spin cytochrome P-450scc

400 MHz ¹H NMR of ferric low-spin cytochrome P-450_scc purified from bovine adrenal cortex was measured for the first time. As compared with ¹H NMR spectra of low-spin P-450_cam and metMb- mercaptan complexes, paramagnetic shifts of low-spin P-450_scc complexes were more divergent, suggesting that there is a subtle difference in the heme environment between P-450_scc and P-450_cam [1]. The paramagnetic shifts of low-spin complexes of P-450_scc caused by adding nitrogenous inhibitors, aminoglutethimide and metyrapone, were different from those caused by adding an intermediate, 20α-hydroxycholesterol, and a detergent, Tween 20 [2]. The paramagnetic shifts of the metMb-mercaptan complexes were convergent compared with those of ferric low-spin P-450_scc and P-450_cam, suggesting that the electronic character and/or the conformation of the internal thiolate ligand in P-450_scc and P-450_cam are different from those of the external thiolate ligand in metMb-thiolate complexes [3]. The paramagetic shifts of the metMb-mercaptan complexes were dependent on the electron donating factor of the alkyl group of the bound mercaptans [4].Magnetic CD(MCD) spectra of ferric low-spin P-450_scc, rabbit liver P-450 complexes and metMb- mercaptan complexes were also observed at various temperatures. The temperature dependences of the Soret MCD bands for the low-spin P-450 and metMb- mercaptan complexes were decidedly less pronounced than those for the low-spin metMb-CN? or imidazole complexes, suggesting that thiolate ligands markedly influence the Soret MCD band of the ferric low-spin complexes [1]. The suggestion described in [2] implied by the ¹H NMR study was reconfirmed from the temperature dependence study of the Soret MCD [2]. The temperature dependences of the Soret MCD bands for low-spin P-450 complexes having a non-nitrogenous ligand were more pronounced than for those having a nitrogenous ligand.     

Model systems for the coordination chemistry of cytochrome P-450.

Studies on model complexes have supported the presence of a mercaptide as the fifth ligand of cytochrome P-450 monooxygenases. When alcohol or thiol ligands are added to the sixth coordination position of a five-coordinated 4-nitrobenzene thiolate complex of FeIII protoporphyrin IX dimethyl ester chloride low spin complexes with optical and EPR-spectra very similar to cytochrome P-450 are obtained. From a comparison with all ligands of cytochrome P-450 and the model complexes it is concluded that a hard ligands must occupy the sixth coordination position of cytochrome P-450. An imidazole group is less likely, also in view of the ligand field parameters. The significance of the fifth and sixth ligand of cytochrome P-450 is discussed with respect to the monooxygenase mechanism.     

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces changes in the heme spin state of microsomal cytochrome P-450

In vitro studies on the nature of interaction of the neurotoxin MPTP with hepatic microsomal cytochrome P-450 were carried out. Spectral perturbation studies showed nitrogenous ligand type binding between MPTP and cytochrome P-450 with a peak at 423 nm and a broad trough at 400 nm. Scatchard analysis of MPTP-cytochrome P-450 binding suggested that MPTP binds to at least 2 species of cytochrome P-450--a high affinity binding species with an apparent spectral dissociation constant (Ks) of 372 microM and a low affinity species with Ks of 37.6 mM. EPR studies confirmed that MPTP is a type II substrate for the forms of cytochrome P-450 with which it interacts and causes a shift from the high spin state of cytochrome P-450 to the low spin state. MPTP is, thus, likely to be an effective inhibitor of cytochrome P-450.     

Spectroscopic investigations of ferric cytochrome P-450-CAM ligand complexes. Identification of the ligand trans to cysteinate in the native enzyme

An extensive series of ligand complexes of ferric cytochrome P-450-CAM has been examined by UV-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy in an attempt to identify the ligand trans to cysteinate in the six-coordinate resting state of the enzyme. Thus, the ligands used have been chosen to serve as models for coordination by potential endogenous amino acids and include alcohol, amide and carboxylate oxygen donors, amine, imidazole and indole nitrogen donors and disulfide, thioether, thiol, and thiolate sulfur donors. As this investigation has been by nature an empirical one, the conclusions are strengthened by the concurrent use of three different spectroscopic techniques. All of the complexes formed except those resulting from thiolate addition display spectroscopic properties that are broadly similar to those of low spin, six-coordinate P-450. Of the sulfur donor adducts, disulfide and thioether-bound P-450 have properties that are different enough in detail to distinguish them from native P-450. While the spectral features of the thiol-bound species and of low spin ferric P-450 are alike, the former are pH dependent due to interconversion to bound thiolate, whereas the latter display essentially no spectral changes with pH. Of the oxygen donor complexes, all but carboxylate have spectra that very closely match those of the resting enzyme. Adducts formed with most nitrogenous ligands, including several imidazole derivatives, exhibit spectra that are sufficiently different from native P-450 to exclude them as candidates for the sixth ligand. Interestingly, the spectral properties of a complex formed with an imidazole derivative having a bulky electron-withdrawing substituent in the alpha position are comparable to those native P-450 except for the line shape of the EPR spectrum. Previously published theoretical work suggests that the spectral differences seen between this imidazole derivative and the other examined are electronic and not steric in origin. As no similar electronic mechanism exists for the protein to reduce the electron-donating ability or histidine, it is felt that coordination of histidine in the sixth position of P-450 can be ruled out. In conclusion, close examination of all spectral data reveals that amino acid analog adducts of P-450-CAM with amides and, in particular, alcohols, produce spectra that almost exactly duplicate those of native P-450 and suggests that the ligand trans to cysteinate in the six-coordinate ferric enzyme has an oxygen donor atom.     

Low- and ultralow-temperature magnetic circular dichroism studies of reduced cytochromes P-450-LM2 and P-420-LM2 and of photo-products of their co-complexes. The spin-state and axial ligation of heme iron

MCD spectra of reduced cytochromes P-450 and P-420 have been recorded in the spectral region 350-800 nm at temperatures 4.2-290 K and were compared with the respective low-temperature photolysed CO-complexes at 4.2 K. The MCD data are consistent with the suggestions that: the heme iron is high-spin in the reduced proteins and in the photolysed species; mercaptide is the protein-derived ligand of the heme iron in the reduced cytochrome P-450, as well as in its CO-complex; imidazole of histidine is the fifth ligand of the heme iron both in the reduced P-420 and its CO-complex; structural changes in the heme iron coordination sphere occur at CO-binding.     

Binding of benzo[a]pyrene by purified cytochrome P-450c

Benzo[a]pyrene (BP) fluorescence-emission intensities in phospholipid micelles are quantitatively described over a broad range of lipid and BP concentrations by excitation that is linearly dependent upon BP concentration and an offsetting excimer quenching that is dependent upon the square of the BP concentration. The fluorescence of BP is quenched by the presence of cytochrome P-450c in proportion to the concentration of the cytochrome in the micelles and in accord with stoichiometric complex formation. Parallel optical titrations indicate a change in spin state of P-450c to a predominantly high-spin state that correlates directly with the percentage fluorescence quenching of complexed BP. Neither change occurs with five other purified forms of rat liver P-450 that have low activity in BP metabolism. N-Octylamine, a ligand that binds to the heme of P-450, competitively inhibits both the spin-state changes and the fluorescence quenching in equal proportion. The Kd for the interaction of BP with P-450c is exceptionally low (10 nM) relative to the Km for monooxygenation (ca. 1 microM). Decreasing the concentration of either dilauroylphosphatidylcholine or dioleoylphosphatidylcholine concomitantly increases the high-spin state (from 30% to 80%) of fully complexed P-450c and the fluorescence quenching (50-100%) of the complexed BP (half-maximal at 80 micrograms of lipid/mL). It is concluded that spin state and fluorescence quenching both reflect the same changes in the interaction of the BP with the P-450 heme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversible transfer of heme between different molecular species of microsome-bound cytochrome P-450 in rat liver

Incorporation of newly synthesized heme into microsome-bound cytochrome P-450 in rat liver was not affected by cycloheximide administration to the animals, indicating that the heme incorporation into cytochrome P-450 is not tightly coupled with the synthesis of the apo-cytochrome. When the heme of microsomal cytochrome P-450 had been labeled in vivo with delta-[14C]aminolevulinic acid, and then the animals were treated with phenobarbital (PB) or 3-methylcholanthrene (MC), PB-induced or MC-induced form of cytochrome P-450 was found to contain labeled heme derived from preexistent cytochrome P-450. These observations indicated that the heme of microsome-bound cytochrome P-450 is not tightly associated with the protein portion, and exchanges reversibly between different molecular species of cytochrome P-450 in vivo.     

Cytochrome P-450 heme and the regulation of hepatic heme oxygenase activity

The degradation of cytochrome P-450 heme in the liver has been studied by a new approach. In rats, hepatic heme was labeled by administration of a tracer pulse of [5-¹⁴C]δ-aminolevulinic acid (ALA), and its degradation was analyzed in terms of labeled carbon monoxide (¹⁴CO) excretion, which is a specific degradation product of the labeled heme. Within minutes after administration of [5-¹⁴C]ALA, 14CO was detectable and increased after 2 h to an “early peak,” reflecting the elimination of labeled heme from a rapidly turning over pool in the liver. Beyond the early peak, the rate of ¹⁴CO production decreased in a log-linear manner, consistent with the degradation of heme in stable hepatic hemoproteins. From the rate at which ¹⁴CO production declined during this phase, from the predominant labeling of cytochrome P-450 heme by the administered [5-¹⁴C]ALA and from the known turnover characteristics of this hemoprotein in the liver, it could be inferred that production of ¹⁴CO—between 16 and 30 h after administration of labeled ALA—largely reflected degradation of cytochrome P-450 heme. This approach, which permits serial measurements in a single animal, was used to study the effect on cytochrome P-450 heme of administered heme or endotoxin, both of which are potent stimulators of hepatic heme oxygenase activity. Both of these substances caused marked acceleration of the degradation of cytochrome P-450 heme, the effect occurring over the same dose range as that for stimulation of hepatic heme oxygenase. The findings suggest that stimulation of this enzyme activity in the liver is closely related to the rate of degradation of cytochrome P-450 heme.     

Incorporation of heme to microsomal cytochrome P-450 in the absence of protein biosynthesis

Determination of the heme and protein portions of phenobarbital (PB)-inducible and 3-methylcholanthrene inducible forms of cytochrome P-450, P-450(PB-1), and P-450(MC-1), in the liver microsomes of drug-treated animals indicated the presence of 20-30% of apo-cytochrome P-450 in both cases. Inhibition of protein synthesis by cycloheximide injection to the rats did not significantly inhibit the incorporation of delta-amino[14C]levulinic acid (ALA) into the heme of P-450(PB-1) or P-450(MC-1) in the liver, indicating that the heme incorporation into microsomal cytochrome P-450 is not tightly coupled with the synthesis of the apo-cytochrome. When heme-labeled cytosol prepared from [14C]ALA-injected rats was incubated with non-radioactive microsomes in vitro, a significant amount of labeled heme was incorporated into microsomal P-450(PB-1), whereas the incorporation into P-450(MC-1) was much less. The in vitro transfer of heme from cytosol to microsome-bound cytochrome P-450 was stimulated by the addition of an NADPH-generating system to the incubation mixtures, and inhibited when the microsomes were solubilized with sodium cholate and Emulgen-913. Although the in vitro incubation of heme-labeled microsomes with non-radioactive cytosol resulted in some release of labeled heme from the microsomes, no reversible transfer of heme between cytochrome P-450 molecules bound to separate microsomal vesicles was detected when heme-labeled microsomes were incubated with non-radioactive microsomes in the presence and absence of cytosol.     

Protein-protein interactions in microsomal cytochrome P-450 isozyme LM2 and their effect on substrate binding

The effects of protein-protein interactions and substrate binding on the structure of the active site of rabbit liver microsomal cytochrome P-450 LM2 have been analyzed by resonance Raman spectroscopy of the monomeric and oligomeric protein in solution. Also H2O2-dependent catalytic activities of the two states have been compared. The two vinyl substituents of the heme exhibit different orientations, as indicated by the frequencies and intensities of their stretching vibrations. One group lies in the plane of the heme and remains unchanged in the two states of cytochrome P-450 LM2, the other is tilted out of the plane. The tilting angle in oligomers was smaller than in monomers. These vinyl stretching modes together with some porphyrin modes, were found to be sensitive indicators of the quaternary structure and of substrate binding. In both the oligomer and the monomer, substrate binding causes changes of the relative intensities of some porphyrin modes and the vinyl stretching vibrations which may reflect modifications of the electronic transitions due to hydrophobic interactions between the bound substrate and the heme. In contrast to the monomeric cytochrome P-450 LM2, benzphetamine binding to the oligomers of this isozyme additionally produces a shift of the spin-state equilibrium. This indicates that in the oligomer the substrate-binding pocket is converted by protein-protein interaction to a structure that forces substrates to interfere with the sixth ligands, inducing an increase of the five-coordinated high-spin configuration. In the monomer the substrate-binding pocket can accommodate benzphetamine without affecting the spin state. Binding of imidazole to the monomeric and oligomeric cytochrome P-450 LM2 produces essentially the same resonance Raman spectra. Apparently the replacement of the native sixth ligand by imidazole disturbs the structure of the active site in such a way that it becomes insensitive to protein-protein interactions. H2O2-dependent N-demethylation of benzphetamine and aniline p-hydroxylation by cytochrome P-450 LM2 did not depend on its state of aggregation.     

Electron spin echo envelope modulation spectroscopic study of iron-nitrogen interactions in myoglobin hydroxide and Fe(III) tetraphenylporphyrin models.

The electron-nuclear coupling in low-spin iron complexes including myoglobin hydroxide (MbOH) and two related model compounds, Fe(III) tetraphenylporphyrin(pyridine)(OR-) (R = H or CH3) and Fe(III) tetraphenylporphyrin(butylamine)(OR-) was investigated using electron spin echo envelope modulation (ESEEM) spectroscopy. The assignment of frequency components in ESEEM spectra was accomplished through the use of nitrogen isotopic substitution wherever necessary. For example, the proximal imidazole coupling in MbOH was investigated without interference from the contributions of porphyrin 14N nuclei after substitution of the heme in native Mb with 15N-labeled heme. Computer simulation of spectra using angle selected techniques enabled the assignment of parameters describing the hyperfine and quadrupole interactions for axially bound nitrogen of imidazole in MbOH, of axial pyridine and butylamine in the models, and for the porphyrin nitrogens of the heme in native MbOH. The isotropic component of axial nitrogen hyperfine interactions exhibits a trend from 5 to 4 MHz, with imidazole (MbOH) greater than pyridine greater than amine. The nuclear quadrupole interaction coupling constant e2Qq was near 2 MHz for all nitrogens in these complexes. The Qzz axis of the nuclear quadrupole interaction tensor for the proximal imidazole nitrogen in MbOH was found to be aligned near gz (gmax) in MbOH, suggesting that gz is near the heme normal. A crystal field analysis, that allows a calculation of rhombic and axial splittings for the d orbitals of the t2g set in a low-spin heme complex, based on the g tensor assignment gz greater than gy greater than gx, yielded results that are consistent with the poor pi-acceptor properties expected for the closed shell oxygen atom of the hydroxide ligand in MbOH. A discussion is presented of the unusual results reported in a linear electric field effect in EPR (LEFE) study of MbOH published previously [Mims, W. B., & Peisach, J. (1976) J. Chem. Phys. 64, 1074-1091].     

H93G myoglobin cavity mutant as versatile template for modeling heme proteins: magnetic circular dichroism studies of thiolate- and imidazole-ligated complexes

Recent ligand binding and spectroscopic investigations of the myoglobin H93G cavity mutant are reviewed, revealing it to be a versatile template for the preparation of model heme complexes of defined structure. The H93G myoglobin cavity mutant is shown to be capable of forming mixed ligand adducts because of the difference in accessibility of the two sides of the ferric heme iron. With imidazole bound in the proximal cavity, H93G myoglobin also forms reasonably stable oxyferrous and oxoferryl derivatives, thereby providing a potential system to use for the study of such complexes with proximal ligands other than imidazole. In addition, thiolate-ligated ferric H93G derivatives are described that serve as spectroscopic models for the high-spin ferric state of cytochrome P450. All of the complexes described are characterized with magnetic circular dichroism spectroscopy, and they are compared to the appropriate derivatives of native myoglobin and P450.     

Spectral characterization of diarylpropane oxygenase, a novel peroxide-dependent, lignin-degrading heme enzyme

Diarylpropane oxygenase, an H2O2-dependent lignin-degrading enzyme from the basidiomycete fungus Phanerochaete chrysosporium, catalyzes the oxygenation of various lignin model compounds with incorporation of a single atom of dioxygen (O2). Diarylpropane oxygenase is also capable of oxidizing some alcohols to aldehydes and/or ketones. This enzyme (Mr = 41,000) contains a single iron protoporphyrin IX prosthetic group. Previous studies revealed that the Soret maximum of the ferrous-CO complex of diarylpropane oxygenase is at approximately 420 nm, as in ferrous-CO myoglobin (Mb), and not like the approximately 450 nm absorption of the CO complex of the ubiquitous heme monooxygenase, cytochrome P-450. This spectral difference between two functionally similar heme enzymes is of interest. To elucidate the structural requirements for heme iron-based oxygenase reactions, we have compared the electronic absorption, EPR, and resonance Raman (RR) spectral properties of diarylpropane oxygenase with those of other heme proteins and enzymes of known axial ligation. The absorption spectra of native (ferric), cyano, and ferrous diarylpropane oxygenase closely resemble those of the analogous myoglobin complexes. The EPR g values of native diarylpropane oxygenase, 5.83 and 1.99, also agree well with those of aquometMb. The RR spectra of ferric diarylpropane oxygenase have their spin- and oxidation-state marker bands at frequencies analogous to those of aquometMb and indicate a high-spin, hexacoordinate ferric iron. The RR spectra of ferrous diarylpropane oxygenase have frequencies analogous to those of deoxy-Mb that suggest a high-spin, pentacoordinate Fe(II) in the reduced form. The RR spectra of both ferric and ferrous diarylpropane oxygenase are less similar to those of horseradish peroxidase, catalase, or cytochrome c peroxidase and are clearly distinct from those of P-450. These observations suggest that the fifth ligand to the heme iron of diarylpropane oxygenase is a neutral histidine and that the iron environment must resemble that of the oxygen transport protein, myoglobin, rather than that of the peroxidases, catalase, or P-450. Given the functional similarity between diarylpropane oxygenase and P-450, this work implies that the mechanism of oxygen insertion for the two systems is different.     

Hydrogen bonding to the bound dioxygen in oxy cobaltous myoglobin reduces the superhyperfine coupling to the proximal histidine.

Electron spin echo envelope modulation (ESEEM) spectroscopy was used to study the electron-nuclear coupling in two oxygenated cobalt-substituted hemoproteins, myoglobin (oxyCoMb) and a monomeric hemoglobin from Glycera dibranchiata (oxyCoHbgly). The modulation frequency components in ESEEM spectra of both proteins arose from the coupling to the N epsilon of the proximal histidyl imidazole. The hyperfine and quadrupole coupling parameters for these two nitrogens, calculated by computer spectral simulation, are Aiso = 2.46 MHz, e2qQ = 2.15 MHz, and eta = 0.4 for oxyCoMb and Aiso = 3.70 MHz, e2qQ = 2.70 MHz, and eta = 0.5 for oxyCoHbgly. A hyperfine coupling of 0.6 MHz, found for oxyCoMb in D2O but not for oxyCoHbgly in D2O, was assigned to the coupling to a deuteron that is hydrogen-bonded to the O2 ligand in oxyCoMb. This hydrogen bonding is believed to be responsible for the reduction in hyperfine and nuclear quadrupole coupling to the proximal histidyl imidazole N epsilon in oxyCoMb. A molecular orbital model for O2 adducts of cobaltous compounds [Tovrog et al. (1976) J. Am. Chem. Soc. 98, 5144] was used to understand the hydrogen bond-induced reduction in 14N superhyperfine coupling in oxyCoMb.