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Cytochrome bo from Escherichia coli: reaction of the oxidized enzyme with hydrogen peroxide.

ABSTRACT: Oxidized cytochrome bo reacts rapidly with micromolar concentrations of H2O2 to form a single derivative. The electronic absorption spectrum of this compound differs from that of the oxidized form of the enzyme reported by this laboratory [Watmough, Cheesman, Gennis, Greenwood and Thomson (1993) FEBS Lett. 319, 151-154]. It is characterized by a Soret maximum at 411 nm, increased absorbance at 555 nm, and reduced intensity at 624 nm. The apparent dissociation constant for this process is of the order of 4 x 10(-6) M, and the bimolecular rate constant for the formation of the new compound is (1.25-1.7) x 10(3) M-1.s-1. Electronic absorption difference spectroscopy shows this product to be identical with the compound formed from the reaction of the mixed-valence form of the enzyme with dioxygen. Investigation of this compound by room-temperature magnetic c.d. spectroscopy shows haem o to be neither high-spin nor low-spin ferric, but to have a spectrum characteristic of an oxyferryl species. There is no evidence for oxidation of the porphyrin ring. Therefore the binuclear centre of this species must consist of an oxyferryl haem (S = 1) coupled to a Cu(II) ion (S = 1/2) to form a new paramagnetic centre. The reaction was also followed by X-band e.p.r. spectroscopy, and this showed the disappearance in parallel with the formation of the oxyferryl species, of the broad g = 3.7, signal which arises from the weakly coupled binuclear centre in the oxidized enzyme. Since no new e.p.r.-detectable paramagnetic species were observed, the Cu(II) ion is presumed to be coupled to another paramagnet, possibly an organic radical. There is no evidence in the electronic absorption spectrum to indicate further reaction of cytochrome bo with H2O2 to form a second species. We argue that the circumstances of formation of this oxyferryl species are the same as those for the P form of cytochrome c oxidase, a species often regarded as containing a bound peroxide ion. The implications of these observations for the reaction mechanism of haem-copper terminal oxidases are discussed.

SUBMITTER: Watmough NJ

PROVIDER: S-EPMC1138186 | biostudies-other | 1994 Jun

REPOSITORIES: biostudies-other

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Project description:Inner membranes were prepared from Escherichia coli strain RG 145, which is deficient in cytochrome bd, but overexpresses cytochrome bo [Au and Gennis (1987) J. Bacteriol. 169, 3237-3242]. The latter was purified 7-fold by extracting the membranes with octyl beta-D-glucopyranoside, followed by chromatography on DEAE-Sepharose, yielding 150 mg of protein/150 g wet weight of cells. Optical e.p.r. and low-temperature m.c.d. (magnetic circular dichroism) spectroscopies were used to investigate the nature of the protein ligands to the two haems in cytochrome bo from E. coli. Low-spin ferric haem b, the origin of a rhombic e.p.r. spectrum with g = 2.98, 2.26 and 1.50, gives rise to a charge-transfer band in the near-i.r. m.c.d. spectrum at 1622 nm. It is therefore concluded that haem b is co-ordinated by two histidine residues. The low-temperature m.c.d. spectrum of dithionite-reduced cytochrome bo comprises bands due both to low-spin ferrous haem b and to high-spin ferrous haem o. The bands arising from haem o show a direct correspondence with those in the m.c.d. spectrum of five-co-ordinate histidine-ligated ferrous haems such as myoglobin, implying that the protein residue liganding haem o is also histidine. This assignment was confirmed by measuring the e.p.r. spectrum of the nitric oxide derivative of fully reduced cytochrome bo. This showed a rhombic spectrum with g = 2.098, 2.008 and 1.987, and nuclear hyperfine splitting consistent with the co-ordination of ferrous haem by NO and histidine. The hyperfine splittings observed were 1.95 +/- 0.05 mT for the 14N of the NO ligand and 0.75 +/- 0.05 mT for the 14N of the proximal histidine. The e.p.r. spectrum of some samples of oxidized cytochrome bo show, at temperatures below 15 K, broad signals at g = 7.6, 3.6 and 2.8, and other preparations in the presence of glycerol yield signals at g = 10.8, 3.2 and 2.6. These signals, which are abolished by the addition of cyanide, are assigned to the binuclear centre, cytochrome o-CuB, suggesting that the binuclear site may display heterogeneity. Carbon monoxide reacts with the reduced enzyme with a stoichiometry of 1:1, and the dissociation constant for this reaction was determined to be 1.7 x 10(-6)M. The second-order rate constants for this reaction were measured and shown to be similar to those determined for bovine cytochrome aa3 [Gibson and Greenwood (1963) Biochem. J. 86, 541-554].

Project description:The R481 residue of cytochrome bo(3) ubiquinol oxidase from E. coli is highly conserved in the heme-copper oxidase superfamily. It has been postulated to serve as part of a proton loading site that regulates proton translocation across the protein matrix of the enzyme. Along these lines, proton pumping efficiency has been demonstrated to be abolished in many R481 mutants. However, R481Q in bo(3) from E. coli has been shown to be fully functional, implying that the positive charge of the arginine is not required for proton translocation [ Puustinen , A. and Wikstrom , M. ( 1999 ) Proc. Natl. Acad. Sci. U.S.A. 96 , 35 - 37 ]. In an effort to delineate the structural role of R481 in the bo(3) oxidase, we used resonance Raman spectroscopy to compare the nonfunctional R481L mutant and the functional R481Q mutant, to the wild type protein. Resonance Raman data of the oxidized and reduced forms of the R481L mutant indicate that the mutation introduces changes to the heme o(3) coordination state, reflecting a change in position and/or coordination of the Cu(B) located on the distal side of heme o(3), although it is approximately 10 A away from R481. In the reduced-CO adduct of R481L, the frequencies of the Fe-CO and C-O stretching modes indicate that, unlike the wild type protein, the Cu(B) is no longer close to the heme-bound CO. In contrast, resonance Raman data obtained from the various oxidation and ligation states of the R481Q mutant are similar to those of the wild type protein, except that the mutation causes an enhancement of the relative intensity of the beta conformer of the CO-adduct, indicating a shift in the equilibrium between the alpha and beta conformers. The current findings, together with crystallographic structural data of heme-copper oxidases, indicate that R481 plays a keystone role in stabilizing the functional structure of the Cu(B) site through a hydrogen bonding network involving ordered water molecules. The implications of these data on the proton translocation mechanism are considered.

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Cytochrome bo from Escherichia coli: reaction of the oxidized enzyme with hydrogen peroxide.

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