title: A rapid-scan spectrometric and stopped-flow study of compound I and compound II of Pseudomonas cytochrome c peroxidase.
abstract: A quantitative yield of half-reduced (ferrous-ferric) cytochrome c peroxidase from Pseudomonas aeruginosa has been obtained by using either ascorbate or NADH as reductant of the resting (ferric-ferric) enzyme along with phenazine methosulfate as mediator. The formation of Compounds I and II from the half-reduced enzyme and hydrogen peroxide has been studied at 25 degrees C using rapid-scan spectrometry and stopped-flow measurements. The spectra of Compound I in the Soret and visible regions were recorded within 5 ms after mixing the half-reduced enzyme with H2O2. The spectrum of the primary compound at the Soret region had a maximum at 414 nm, and in the visible region at 528 and 556 nm. The spectrum of Compound I showed no bands in the 650-nm region, excluding the possibility of a pi-cation radical being part of the catalytic mechanism. Compound I was stable for at least 12 s when no reducing equivalents were present. In the presence of reduced azurin, half-reduced enzyme reacted with H2O2 to form Compound II within 50 ms. The spectrum of Compound II had a Soret maximum at 411 nm. In the visible region the Compound II spectrum was close to that of the totally oxidized, resting enzyme form. In the presence of excess azurin, Compound II was converted rapidly to the half-reduced enzyme form. The kinetics of Compound I formation was also followed with peracetic acid, ethylhydroperoxide, and m-chloroperbenzoic acid as electron acceptors. The rate constants of these reactions are diminished compared to that of hydrogen peroxide, indicating a closed structure for the heme pocket of the enzyme.
authors.forename: M
authors.initials: M
authors.lastname: Rönnberg
authors.forename: A M
authors.initials: AM
authors.lastname: Lambeir
authors.forename: N
authors.initials: N
authors.lastname: Ellfolk
authors.forename: H B
authors.initials: HB
authors.lastname: Dunford
mesh_terms: D055598:Chemical Phenomena; D002621:Chemistry; D003578:Cytochrome-c Peroxidase / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D013053:Spectrophotometry / Q000379:methods
chemical_list: D010544:Peroxidases; D003578:Cytochrome-c Peroxidase