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 determ ination was developed. This was combined with a newly designed microci rculating system and was successfully applied to study intermediates i n the reaction of bovine oxidized cytochrome c oxidase (CcO) with hydr ogen peroxide under steady state conditions at ambient temperatures. M easurements with this device made it possible to correlate directly th e species defined in terms of the visible absorption characteristics w ith specific Raman bands. The ''607 nm'' form of the enzyme obtained w ith (H2O2)-O-16 gave an oxygen isotope sensitive band at 804 cm(-1) (7 69 cm(-1) with (H2O2)-O-18) in the Soret excited resonance Raman (RR) spectrum. Its frequency and isotopic frequency shifts are exactly the same as those observed previously with 607 nm excitation in nonsimulta neous measurements for the 607 nm form, for which the presence of an o xoiron heme was demonstrated. The so-called ''580 nm'' form of the enz yme obtained with (H2O2)-O-16 gave the main oxygen isotope sensitive b and at 785 cm(-1) (750 cm(-1) with (H2O2)-O-18) but appeared to consis t of multiple species. This band was assigned to the Fe-IV=O stretchin g 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 (H2O2)-O-18). Similar to the case of dioxygen reaction. Tempo ral 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 th e two species. The RR spectra in the higher frequency region of the 60 7 nm and 580 nm forms excited ar 427 nm were quite alike and did nor s upport the formation of a porphyrin pi-cation radical.