Oxidase reaction of cytochrome cd(1) from Paracoccus pantotrophus

Cytochrome cd(1) (cd(1)NIR) from Paracoccus pantotrophus, which is both a n itrite reductase and an oxidase, was reduced by ascorbate plus hexaaminerut henium(III) chloride on a relatively slow time scale (hours required for co mplete reduction). Visible absorption spectroscopy showed that mixing of as corbate-reduced enzyme with oxygen at pH = 6.0 resulted in the rapid oxidat ion of both types of heme center in the enzyme with a linear dependence on oxygen concentration. Subsequent changes on a longer time scale reflected t he formation and decay of partially reduced oxygen species bound to the d(1 ) heme iron. Parallel freeze-quench experiments allowed the X-band electron paramagnetic resonance (EPR) spectrum of the enzyme to be recorded at vari ous times after mixing with oxygen. On the same millisecond time scale that simultaneous oxidation of both heme centers was seen in the optical experi ments, two new EPR signals were observed. Both of these are assigned to oxi dized heme c and resemble signals from the cytochrome c domain of a "semi-a po" form of the enzyme for which histidine/methionine coordination was demo nstrated spectroscopically. These observations suggests that structural cha nges take around the heme c center that lead to either histidine/methionine axial ligation or a different stereochemistry of bis-histidine axial ligat ion than that found in the as prepared enzyme. At this stage in the reactio n no EPR signal could be ascribed to Fe(III) d(1) heme. Rather, a radical s pecies, which is tentatively assigned to an amino acid radical proximal to the d(1) heme iron in the Fe(IV)-oxo state, was seen. The kinetics of decay of this radical species match the generation of a new form of the Fe(III) d(1) heme, probably representing an OH--bound species. This sequence of eve nts is interpreted in terms of a concerted two-electron reduction of oxygen to bound peroxide, which is immediately cleaved to yield water and an Fe(I V)-oxo species plus the radical. Two electrons from ascorbate are subsequen tly transferred to the d(1) heme active site via heme c to reduce both the radical and the Fe(IV)-oxo species to Fe(III)-OH- for completion of a catal ytic cycle.