The fully oxidized state of cytochrome c peroxidase (CcP), called ES, contains two oxidizing equivalents, one as an oxyferryl heme and the other as an organic radical on an amino acid residue. The unusual electron paramagnetic resonance spectrum of ES has been shown to be due to a weak, distributed exchange coupling between the two paramagnetic redox centers (Houseman, A.

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We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type [2Fe-2S] cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) [15N]histidine in a 14N background; (4) [14N]histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske [2Fe-2S] center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type [2Fe-2S] centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier Mössbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled [Fe2+ (S = 2), Fe3+ (S = 5/2)] pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.

EPR, 35-GHz ENDOR, and X-ray absorption (XAS) spectroscopic studies of H2 binding in hydrogenase from Thiocapsa roseopersicina are reported. These studies involve spectra taken on an active form of the enzyme that displays an EPR signal designated Ni-C. This form of the enzyme is light sensitive and is converted to a photoproduct that exhibits a unique EPR signal, Ni-L. 'H-ENDOR spectra reveal two sets of protons that interact strongly with the EPR-active center in Ni-C. The first proton set (HI) is solvent exchangeable, originates from dihydrogen, and has a value of A ( H 1 ) -20 MHz. The second proton set (H2) is not solvent exchangeable, has a coupling constant of A(H2) -12 MHz, and is assigned to one or more cysteine P-CH2 protons. Upon irradiation and conversion of the EPR signal to Ni-L, the exchangeable HI proton set is no longer observed. Upon annealing and regeneration of Ni-C, the feature associated with the H I proton set is again observed. This is direct evidence that the photoprocess and annealing involve dissociation of a hydrogenic proton and recombination. XAS spectra taken on samples poised to maximize Ni-C and Ni-L do not reveal any significant structural difference between the Ni sites.

Continuous wave electron nuclear double resonance (CW ENDOR) spectra of [delta-15N,epsilon(-14)N]histidine-labeled phthalate dioxygenase (PDO) from Pseudomonas cepacia were recorded and found to be virtually identical to those previously recorded from [delta,epsilon-15N2]histidine-labeled protein [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871]. Thus, the two histidine residues, previously shown to ligate one of the irons in the cluster [cf. Gurbiel et al. 1989)], both coordinate the metal at the N(delta) position of their imidazole rings. Pulsed ENDOR studies showed that the "remote", noncoordinating nitrogen of the histidine imidazole ring could be observed from the Rieske protein in a sample of Rhodobacter capsulatus cytochrome bc1 complex uniformly labeled with 15N but not in a sample of PDO labeled with [delta-15N,epsilon-14N]histidine, but this atom was easily observed with a sample of Rh. capsulatus cytochrome bc1 complex that had been uniformly labeled with 15N; this confirmed the conclusion from the CW ENDOR studies that ligation is exclusively via N(delta) for both ligands in the PDO center. Modifications in the algorithms previously used to simulate 14N ENDOR spectra permitted us to compute spectra without any constraints on the relative orientation of hyperfine and quadrupole tensors. This new algorithm was used to analyze current and previously published spectra, and slightly different values for the N-Fe-N angle and imidazole ring rotation angles are presented [cf. Gurbiel et al. (1989) Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., and Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. This analysis has permitted us to refine the proposed structure of the [2Fe-2S] Rieske-type cluster and rationalize some of the properties of these novel centers. Although the spectra of cytochrome bc1 complex from Rh. capsulatus are of somewhat lower resolution than those obtained with samples of PDO, our analysis nevertheless permits the conclusion that the geometry of the cluster is essentially the same for all Rieske and Rieske-type proteins. Structural constraints inferred from the spectroscopic results permitted us to apply the principles of distance geometry to arrive at possible three-dimensional models of the active site structure of Rieske protein from Rh. capsulatus. Results from this test case indicate that similar procedures should be generally useful in metalloprotein systems. We also recorded the pulsed and CW ENDOR spectra of 57Fe-labeled PDO, and the resulting data were used to derive the full hyperfine tensors for both Fe(III) and Fe(II) ions, including their orientations relative to the g tensor. The A tensor of the ferric ion is nominally isotropic, while the A tensor of the ferrous ion is axial, having A(parallel) > A(perpendicular); both tensors are coincident with the observed g tensor, with A(parallel) of the ferrous ion lying along the maximum g-value, g1. These results were examined using re...

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Electron nuclear double resonance (ENDOR) experiments were performed on 14N (natural abundance) and 15N-enriched iron-sulfur Rieske protein in the ubiquinol-cytochrome c2 oxidoreductase from Rhodobactor capsulatus. The experiments proved that two distinct nitrogenous ligands, histidines, are undoubtedly ligated to the Rieske [2Fe-2S] center. The calculations of hyperfine tensors give values similar but not identical to those of the Rieske-type cluster in phthalate dioxygenase of Pseudomonas cepacia and suggest a slightly different geometry of the iron-sulfur cluster in the two proteins.

The binding of several exogenous molecules at the active site of the soluble methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) is described. The interaction of methanol, the product of methane hydroxylation, with the mixed-valent FeIIFeIII form of the enzyme induces a change in the EPR spectrum of the unmodified protein from g = 1.940, 1.865, and 1.740 to one with g 1 = 1.963 and g 2 = 1.862. This spectral change arises from the coordination of methanol to the diiron center, as demonstrated by electron nuclear double resonance (ENDOR) spectroscopic studies of mixed-valent MMOH prepared with CD3OH added to a DMSO-treated sample, which displayed a 2H interaction with an isotropic hyperfine splitting of ∼0.5 MHz. A detailed analysis indicates that methanol binds to the ferrous iron without displacing a terminally bound water molecule identified previously. Acetate labeled with 13C at the carboxylate carbon atom also gives rise to ENDOR signals, whereas no such signals are seen when the label is at the methyl carbon atom or when the methyl group is deuterated. These results suggest that acetate is oriented in the active site with its carboxylate group pointing toward the iron center, confirming a previous interpretation of X-ray crystallographic experiments. At the MMOH diiron core, four coordination positions that are not usually occupied by endogenous protein ligands are identified which can simultaneously accommodate DMSO, methanol, and water. The implications of these results for the catalytic mechanism of the enzyme are discussed.