RESONANCE RAMAN ABSORPTION CHARACTERIZATION OF THE OXO INTERMEDIATES OF CYTOCHROME-C-OXIDASE GENERATED IN ITS REACTION WITH HYDROGEN-PEROXIDE - PH AND H2O2 CONCENTRATION-DEPENDENCE/

Effects of pH and H2O2 concentration on the reaction of cytochrome c o xidase (CcO) with H2O2 were studied with the high-performance Raman/ab sorption simultaneous determination technique reported previously (Pro shlyakov et al., 1996). This reaction generates two intermediates call ed 607- and 580-nm forms, and we found that they show the same oxygen- isotope-sensitive RR bands as those of the intermediates in O-2 reduct ion by CcO. In transient absorption spectra obtained under single turn over conditions, the 607-nm form appeared as the primary intermediate and subsequently the 580-nm and resting forms, suggesting that H2O2 se rves as an oxidant for the resting enzyme but as a reductant for both the 607- and 580-nm forms in the peroxide cycle. The rise rate of abso rption at 607 nm was insensitive to the H2O/D2O exchange, but the deca y was significantly slower in D2O than in H2O. With the microcirculati ng system, each intermediate was maintained at a constant level under steady-stale conditions by supplying H2O2 continuously. In the pH rang e between 7.4 and 10.0, the population of the 607-nm form decreased at higher pH and at higher concentrations of H2O2. The Fe=O stretching ( nu(Fe=O)) frequencies of the oxo heme of the 607-nm form, observed at 804/769 cm(-1) for their (H2O2)-O-16/(H2O2)-O-18 derivatives, were una ltered in this pH range and exhibited a D2O/H2O shift even at pH 10.0. This indicates that the iron-bound oxygen is hydrogen-bonded to a dis tal residue in this pH range. When the 580-nm form is dominant under t he nonsaturating level of H2O2, two other oxygen-isotope-sensitive Ram an bands have been observed at 755/750 cm(-1) and 355/340 cm(-1) at ne utral pH, but the former disappeared above pH 8.5 and the latter above pH 9.0 without significant changes of absorption spectra, suggesting the presence of two separate species in the name of the 580-nm form. H owever, under the saturating concentration of H2O2, these Raman bands were unaltered between pH 7.4 and 10.0. In contrast, in the absence of excess peroxide, no oxygen-isotope-sensitive RR bands were observed d espite dominance of the 580-nm form. The disappearance of these Raman bands demonstrates the occurrence of oxygen exchange between the oxo h eme and bulk water, whose rate surpasses the formation rate of the 580 -nm form al alkaline pH and/or at low H2O2 concentration. Such an oxyg en exchange did not take place in the 607-nm form. Under the identical experimental conditions for generating a particular steady slate, the exchange of H2O with D2O caused significant depopulation of the 580-n m form and concomitant increase of the 607-nm form. This was satisfact orily interpreted in terms of the difference in the decay rate of the 607-nm form between H2O and D2O. Thus, the reduction of the 607-nm for m to the 580-nm form is likely to be a key step of the redox-linked pr oton pumping in the O-2 reduction.