Microcirculating system for simultaneous determination of Raman and absorption spectra of enzymatic reaction intermediates and its application to the reaction of cytochrome c oxidase with hydrogen peroxide.

A new high-performance device for Raman/absorption simultaneous determination was developed. This was combined with a newly designed microcirculating system and was successfully applied to study intermediates in the reaction of bovine oxidized cytochrome c oxidase (CcO) with hydrogen peroxide under steady state conditions at ambient temperatures. Measurements with this device made it possible to correlate directly the species defined in terms of the visible absorption characteristics with specific Raman bands. The "607 nm" form of the enzyme obtained with H2(16)O2 gave an oxygen isotope sensitive band at 804 cm-1 (769 cm-1 with H2(18)O2) in the Soret excited resonance Raman (RR) spectrum. Its frequency and isotope frequency shifts are exactly the same as those observed previously with 607 nm excitation in nonsimultaneous measurements for the 607 nm form, for which the presence of an oxoiron heme was demonstrated. The so-called " 580 nm" form of the enzyme obtained with H2(16)O2 gave the main oxygen isotope sensitive band at 785 cm-1 (750 cm-1 with H2(18)O2) but appeared to consist of multiple species. This band was assigned to the FeIV = O stretching mode of ferryloxo heme on the basis of its isotopic frequency shift. Another oxygen isotope sensitive band was found at 355 cm-1 (340 cm-1 for H2(18)O2), similar to the case of dioxygen reaction. Temporal behavior of this band did not agree with either that of the 804 cm-1 band or that of the 785 cm-1 band but seemed to grow between the two species. The RR spectra in the higher frequency region of the 607 nm and 580 nm forms excited at 427 nm were quite alike and did not support the formation of a porphyrin pi-cation radical.