Raman spectra of heme a, cytochrome oxidase-ligand complexes, and alkaline denatured oxidase

Abstract

We report 441.6 nm excitation resonance Raman spectra of oxidized and reduced monomeric heme a-imidazole, cytochrome oxidase-exogenous ligand complexes in various redox states, and alkaline denatured oxidase. These data show that, in reduced oxidase, the cytochrome a3 Raman spectrum has bands at 215, 364, 1230, and 1670 cm-1 not observed in the cytochrome a spectrum. The appearance of these bands in the reduced cytochrome a3 spectrum is due to interactions between the heme a of cytochrome a3 and its protein environment and not to intrinsic properties of heme a. These interactions are pH sensitive and strongly influence the vibrational spectra of both heme a groups. We assign the 1670-cm-1 band to the heme a formyl substituent and propose that the intensity of the 1670 cm-1 is high for reduced cytochrome a3 because the C==O lies in the porphyrin plane and is very weak for oxidized and reduced cytochrome a, oxidized cytochrome a3, and oxidized and reduced heme a-imidazole because the C==O lies out of the plane. We suggest that movement of the C==O in and out of the plane explains the ligand induced spectral shift in the optical absorption spectrum of reduced cytochrome a3. Finally, we confirm the observation of Adar & Yonetani (private communication) that, under laser illumination, resting oxidase is photoreactive.

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

“…The behavior of the carbonyl stretching mode (1,666 cm-') of the formyl group (21,22) of the cytochrome a3 (see discussion below) is also consistent with complete spectral separation. It is very weak for the CO-bound reduced enzyme with 441.6-nm excitation in Fig.…”

Resonance Raman, Infrared, and EPR Investigation on the Binuclear Site Structure of the Heme-Copper Ubiquinol Oxidases from Acetobacter aceti: Effect of the Heme Peripheral Formyl Group Substitution

“…The formyl CdO stretching bands appeared at 1671 cm -1 in the air-oxidized state (a), at 1668 cm -1 in the reduced state (b), at 1667 cm -1 in the CO-reduced state (c), and at 1651 cm -1 in the CNreduced state (d). Corresponding bands had been assigned for heme a 3 of bovine heart cytochrome c oxidase Salmeen et al, 1978) (Table 1). These frequencies are also very similar to those of the corresponding bands of other heme-copper oxidases ( Table 1).…”

Photoreactions of Cytochrome c Oxidase

“…The direct photoreduction of CcO from a variety of sources and by light of different wavelengths has been reported (21–31). Resonance Raman (RR) spectra of the oxidized enzyme obtained by blue excitation closely resembled those of partially reduced CcO; the spectral changes were found to be dependent on acquisition time and laser power (21–23). Ogura et al (25) identified the principal photoproduct as the reduced low‐spin heme a. Babcock et al (28) concluded that heme a is first photoreduced, followed by heme a 3 , but attributed the photoreduction to flavin impurities as originally proposed by Adar and Yonetani (21).…”

“…External, low potential reductants react with the enzyme to produce the fully reduced enzyme, as well as a mixture of intermediate, equilibrating oxidation states, depending on the number of reducing equivalents delivered to the enzyme (19,20). Similarly, irradiation of the oxidized enzyme with visible or UV light results in autophotoreduction (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31), a process that is poorly understood (30,31).…”

“…The direct photoreduction of CcO from a variety of sources and by light of different wavelengths has been reported (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Resonance Raman (RR) spectra of the oxidized enzyme obtained by blue excitation closely resembled those of partially reduced CcO; the spectral changes were found to be dependent on acquisition time and laser power (21)(22)(23). Ogura eta,!.…”