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The cytochrome bo 3 ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O 2 to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. Two-dimensional electron spin echo envelope modulation has been applied to explore the exchangeable protons involved in hydrogen bonding to the semiquinone by substitution of 1 H 2 O by 2 H 2 O. Three exchangeable protons possessing different isotropic and anisotropic hyperfine couplings were identified. The strength of the hyperfine interaction with one proton suggests a significant covalent O-H binding of carbonyl oxygen O1 that is a characteristic of a neutral radical, an assignment that is also supported by the unusually large hyperfine coupling to the methyl protons. The second proton with a large anisotropic coupling also forms a strong hydrogen bond with a carbonyl oxygen. This second hydrogen bond, which has a significant out-of-plane character, is from an NH 2 or NH nitrogen, probably from an arginine (Arg-71) known to be in the quinone binding site. Assignment of the third exchangeable proton with smaller anisotropic coupling is more ambiguous, but it is clearly not involved in a direct hydrogen bond with either of the carbonyl oxygens. The results support a model that the semiquinone is bound to the protein in a very asymmetric manner by two strong hydrogen bonds from Asp-75 and Arg-71 to the O1 carbonyl, while the O4 carbonyl is not hydrogen-bonded to the protein.Cytochrome bo 3 (cyt bo 3 ) 3 is a terminal oxidase in the aerobic respiratory chain of Escherichia coli. It catalyzes the two-electron oxidation of ubiquinol-8 with a semiquinone (SQ) intermediate in an overall reaction that releases two protons to solution. The available evidence suggests that the cyt bo 3 ubiquinol oxidase has two Q binding sites (1-3): a low affinity site (Q L ) where the substrate quinol is oxidized and the product is released, and a high affinity site where the bound quinone species acts as a conduit for electrons, similar to the role of the Q A site in the bacterial reaction center (3-7, 9). The substrate (QH 2 ) site, referred to as the low affinity site (Q L ), is equilibrated with the quinone pool in the membrane. The high affinity quinone-binding site (Q H ), from which Q is not readily removed, stabilizes the SQ. The quinone bound at the Q H site functions as a tightly bound cofactor. Pulse radiolysis studies (10) have shown that the tightly bound quinone can be rapidly reduced to the semiquinone species and that the tightly bound quinone is essential for rapid electron transfer to heme b. The first order rate constant for the reduction of heme b is 1.5 ϫ 10 3 s Ϫ1 , which is approximately the turnover rate of the enzyme. It is reasonably assumed that there is a rapid (Ͼ10 4 s Ϫ1 ) two-electron reduction of Q H by the bound substrate Q L H 2 , followed by two one-electr...

Recent progress in understanding the Q-cycle mechanism of the bc(1) complex is reviewed. The data strongly support a mechanism in which the Q(o)-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron-sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe-2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Q(o)-site, and the reduced iron-sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c(1) and liberate the H(+). When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O(2) is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b(L) to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b(L) to enhance the rate constant. The acceptor reactions at the Q(i)-site are still controversial, but likely involve a "two-electron gate" in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b(150) phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed. The mechanism discussed is applicable to a monomeric bc(1) complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b(L) hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.

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Burkholderia cepacia AC1100 is able to use the chlorinated compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as the sole source of carbon and energy. CW EPR and one-dimensional ESEEM spectroscopy studies performed earlier indicate the presence of a Rieske-type [2Fe-2S] cluster with two coordinated histidine residues in 2,4,5-T monooxygenase from B. cepacia. This paper describes the application of two-dimensional ESEEM (called HYSCORE) spectroscopy for further characterization of the nitrogens surrounding the reduced Rieske-type cluster. The HYSCORE spectra measured at field positions in the neighborhood of the principal directions of the g tensor contain major contributions from cross-peaks correlating the two double-quantum transitions from each histidine nitrogen. These allow the estimation of the diagonal components of the hyperfine tensors along the principal axes of the g tensor: 4.05, 3.88, and 4.01 MHz (N1) and 4.71, 5.07, and 5.02 MHz (N2). Other spectral features from the histidine nitrogens usually have a much weaker intensity and are occasionally observed in the spectra. HYSCORE measurements have been also performed with the reduced [2Fe-2S] plant ferredoxin-type cluster with four cysteine ligands in a ferredoxin from Porphira umbilicalis, and spectral features produced by the peptide nitrogen are observed. Similar features also appear in the HYSCORE spectra of the Rieske cluster. Systematic differences are observed between 2,4,5-T monooxygenase and published results from related benzene and phthalate dioxygenases that may reflect structural and functional differences in histidine ligation and the nitrogens of nearby amino acids in Riesketype [2Fe-2S] clusters.

Enzymatic N2 reduction proceeds along a reaction pathway comprised of a sequence of intermediate states generated as a dinitrogen bound to the active-site iron-molybdenum cofactor (FeMo-co) of the nitrogenase MoFe protein undergoes six steps of hydrogenation (e−/H+ delivery). There are two competing proposals for the reaction pathway, and they invoke different intermediates. In the ‘Distal’ (D) pathway, a single N of N2 is hydrogenated in three steps until the first NH3 is liberated, then the remaining nitrido-N is hydrogenated three more times to yield the second NH3. In the ‘Alternating’ (A) pathway, the two N’s instead are hydrogenated alternately, with a hydrazine-bound intermediate formed after four steps of hydrogenation and the first NH3 liberated only during the fifth step. A recent combination of X/Q-band EPR and 15N, 1,2H ENDOR measurements suggested that states trapped during turnover of the α-70Ala/α-195Gln MoFe protein with diazene or hydrazine as substrate correspond to a common intermediate (here denoted I) in which FeMo-co binds a substrate-derived [NxHy] moiety, and measurements reported here show that turnover with methyldiazene generates the same intermediate. In the present report we describe X/Q-band EPR and 14/15N, 1,2H ENDOR/-HYSCORE/ESEEM measurements that characterize the N-atom(s) and proton(s) associated with this moiety. The experiments establish that turnover with N2H2, CH3N2H, and N2H4 in fact generates a common intermediate, I, and show that the N-N bond of substrate has been cleaved in I. Analysis of this finding leads us to conclude that nitrogenase reduces N2H2, CH3N2H, and N2H4 via a common A reaction pathway, and that the same is true for N2 itself, with Fe ion(s) providing the site of reaction.

The cytochrome bo 3 ubiquinol oxidase catalyzes the two-electron oxidation of ubiquinol in the cytoplasmic membrane of Escherichia coli, and reduces O 2 to water. This enzyme has a high affinity quinone binding site (Q H ), and the quinone bound to this site acts as a cofactor, necessary for rapid electron transfer from substrate ubiquinol, which binds at a separate site (Q L ), to heme b. Previous pulsed EPR studies have shown that a semiquinone at the Q H site formed during the catalytic cycle is a neutral species, with two strong hydrogen bonds to Asp-75 and either Arg-71 or Gln-101. In the current work, pulsed EPR studies have been extended to two mutants at the Q H site. The D75E mutation has little influence on the catalytic activity, and the pattern of hydrogen bonding is similar to the wild type. In contrast, the D75H mutant is virtually inactive. Pulsed EPR revealed significant structural changes in this mutant. The hydrogen bond to Arg-71 or Gln-101 that is present in both the wild type and D75E mutant oxidases is missing in the D75H mutant. Instead, the D75H has a single, strong hydrogen bond to a histidine, likely His-75. The D75H mutant stabilizes an anionic form of the semiquinone as a result of the altered hydrogen bond network. Either the redistribution of charge density in the semiquinone species, or the altered hydrogen bonding network is responsible for the loss of catalytic function. Escherichia coli cytochrome (cyt)3 bo 3 ubiquinol oxidase catalyzes the two-electron oxidation of ubiquinol and the fourelectron reduction of O 2 to water. The enzyme contains three redox-active metal centers: a low spin heme b, which is involved in quinol oxidation, and the heme o 3 /Cu B bimetallic center, which is the site where O 2 binds and is reduced to water. The ubiquinol oxidation occurs with a semiquinone (SQ) intermediate in an overall reaction that releases two protons to the periplasm. The enzyme contains two Q sites (1-6): a low affinity site (Q L ), which is equilibrated with the quinone pool in the membrane and functions as the substrate (QH 2 ) binding site, and a high affinity (Q H ) site, from which Q is not readily removed, and that stabilizes a SQ (7-10). The quinone bound at the high-affinity site appears to function as a tightly bound cofactor, similar to the Q A site of the reaction centers. Rapid kinetic studies show that the quinone bound at the Q H site is important for rapid reduction of heme b but not for rapid electron transfer from heme b to the heme o 3 /Cu B binuclear center (11,12). The heme o 3 /Cu B site is where O 2 is reduced to H 2 O using the electrons provided by the oxidation of quinol. Hence, the suggested electron transfer sequence is as follows in Reaction 1.The x-ray structure of cytochrome bo 3 (13) does not contain any bound quinone, but site-directed mutagenesis studies (2-4, 13) have identified residues that modulate the properties of the Q H site. The model of the Q H quinone binding site (Fig. 1), including Arg-71, Asp-75, His-98, and Gln-101 residues (13), has...