title: Intramolecular electron transfer in the dihaem cytochrome c peroxidase of Pseudomonas aeruginosa.
abstract: Mutant forms of the enzyme cytochrome c peroxidase from Pseudomonas aeruginosa, in which the peroxidatic haem ligand (H71) and putative haem-bridging amino acid (W94) have been mutated, were produced in an E. coli expression system as a means of investigating possible mechanisms of intramolecular electron transfer within the enzyme. EPR spectroscopy indicated the presence of a high-spin, presumably five-coordinate, peroxidatic haem site in the H71G and H71G/W94A mutants, whilst the W94A mutant apparently retained the normal six-coordinate haem structures. In turnover experiments, these mutants show 55, 4, and <1% activity, respectively, as compared to the wild-type enzyme. The W94A mutant shows essentially no activity in turnover experiments. Circular dichroism spectroscopy indicates no measurable difference in the secondary structure of the H71G mutant from that of the native enzyme, whilst some small differences are observed for the double mutant. Treatment of the oxidised mutant proteins with hydrogen peroxide, in the absence of preactivation or exogenous reductants, yields products that suggest the formation of a tryptophan radical species in the case of the H71 mutant and the production of a porphyrin radical in the case of the double mutant. These results are discussed in terms of the intramolecular electron transfer in this enzyme.
authors.affiliation: School of Biological Sciences, University of Auckland, 3 Symonds Street, Auckland, 1142, New Zealand.
authors.forename: Yong
authors.initials: Y
authors.lastname: Lee
authors.forename: Svetlana
authors.initials: S
authors.lastname: Boycheva
authors.forename: Thomas
authors.initials: T
authors.lastname: Brittain
authors.forename: Peter D W
authors.initials: PD
authors.lastname: Boyd
mesh_terms: D002942:Circular Dichroism; D003578:Cytochrome-c Peroxidase; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004583:Electrons; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000378:metabolism
chemical_list: D006418:Heme; D003578:Cytochrome-c Peroxidase