Resonance Raman spectral isolation of the a and a3 chromophores in cytochrome oxidase

Abstract

Resonance Raman spectra of reduced CO-bound cytochrome oxidase obtained at two different excitation frequencies (441.6 and 413.1 nm) are compared with the spectra of the fully reduced enzyme. In the spectra of the CO-bound complex only the cytochrome a modes are strongly enhanced with 441.6 nm excitation and only the modes of the CO-bound cytochrome a3 heme are strongly enhanced with 413.1-nm excitation. In the fully reduced complex with both excitation frequencies, modes of both cytochrome a and a3 are enhanced. By subtraction we are able to uncover the complete spectrum of the fully reduced ligand-free cytochrome a3 heme. Thus, we report the discrete resonance Raman spectra of cytochromes a2+, a2+3, and a2+3 (CO). The spectra of fully reduced cytochrome a and ligand-free cytochrome a3 are very different especially in the low frequency region. Binding CO to ferrous cytochrome a3 results in electronic structure changes in the heme analogous to those in hemoglobin and myoglobin, from which we conclude that there is nothing electronically unique in the ferrous cytochrome a3 heme to account for its catalytic properties.

Redox-linked transient deprotonation at the binuclear site in the aa 3 -type quinol oxidase from Acidianus ambivalens : Implications for proton translocation

“…A crucial feature that makes the Raman spectrum of aa 3 very useful is a substantial separation between the various lines arising from a and a 3 . The heme modes of the reduced aa 3 oxidase of A. ambivalens in the high-frequency region, which will be discussed in detail elsewhere, match well with those in the bovine enzyme (30). Of particular importance in the present study are the C-O stretching modes of the formyl group (-CHO) of heme a 3 .…”

“…The assignment of the vibrational modes in aa 3 oxidases is well established (30). With the excitation frequency used in this study (413.1 nm), modes from both the low-spin heme a (S ϭ 0) and the high-spin heme a 3 (S ϭ 2) in the reduced enzyme contribute to the Raman spectrum.…”

“…However, a time-dependent change subsequently occurs in the Raman spectrum, involving a slow shift in frequency of the formyl stretching mode of heme a 3 , from 1,660 to 1,667 cm Ϫ1 . Immediately after reduction, the frequency of the a 3 formyl stretching mode (1,660 cm Ϫ1 ) observed in this enzyme is the lowest among the heme a 3 -containing oxidases, where this mode occurs in the 1,662 to 1,669 cm Ϫ1 range (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). The absence of spectral changes in the other modes, including the oxidation state marker, 4 , at 1,355 cm Ϫ1 (data not shown), indicates that the oxidation, coordination, and spin states are not changing as a function of time after reduction in either of the hemes.…”

Cytochrome c oxidase: decay of the primary oxygen intermediate involves direct electron transfer from cytochrome a.

“…Inspection ofthe data indicates that over the time course of the experiments reported here cytochrome a has been significantly oxidized. With a different excitation wavelength for the probe beam, 441.6 nm, which selectively enhances only reduced cytochrome a (24,25), equivalent results were obtained.…”

Resonance Raman quantification of the redox state of cytochromes b and cin‐vivo and in‐vitro

“…The resonance Raman effect selectively enhances the intensity of those molecules that strongly absorb the excitation wavelength. At 532 nm excitation, cytochromes b and c are selectively detected, but not cytochrome a, whose absorption band (Q band) is located around 600 nm [13,14]. Oxidized cytochromes b and c show a specific band at 1638 cm À1 in common.…”