CYTOCHROME BO FROM ESCHERICHIA-COLI - REACTION OF THE OXIDIZED ENZYMEWITH HYDROGEN-PEROXIDE

Oxidized cytochrome bo reacts rapidly with micromolar concentrations o f 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 an d Thomson (1993) FEBS Lett. 319, 151-154]. It is characterized by a So ret maximum at 411 nm, increased absorbance at 555 nm, and reduced int ensity 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) E lectronic absorption difference spectroscopy shows this product to be identical with the compound formed from the reaction of the mixed-vale nce form of the enzyme with dioxygen. Investigation of this compound b y room-temperature magnetic c.d. spectroscopy shows haem o to be neith er high-spin nor low-spin ferric, but to have a spectrum characteristi c of an oxyferryl species. There is no evidence for oxidation of the p orphyrin ring. Therefore the binuclear centre of this species must con sist 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-b and e.p.r. spectroscopy, and this showed the disappearance in parallel with the formation of the oxyferryl species, of the broad g = 3.7, si gnal which arises from the weakly coupled binuclear centre in the oxid ized enzyme. Since no new e.p.r. detectable paramagnetic species were observed, the Cu(II) ion is presumed to be coupled to another paramagn et, possibly an organic radical. There is no evidence in the electroni c absorption spectrum to indicate further reaction of cytochrome bo wi th H2O2 to form a second species. We argue that the circumstances of f ormation of this oxyferryl species are the same as those for the P for m of cytochrome c oxidase, a species often regarded as containing a bo und peroxide ion. The implications of these observations for the react ion mechanism of haem-copper terminal oxidases are discussed.