RESONANCE RAMAN AND OPTICAL SPECTROSCOPIC MONITORING OF HEME-A REDOX STATES IN CYTOCHROME-C-OXIDASE DURING POTENTIOMETRIC TITRATIONS

被引:11
作者
HARMON, PA
HENDLER, RW
LEVIN, IW
机构
[1] NIDDKD,CHEM PHYS LAB,BETHESDA,MD 20892
[2] NHLBI,CELL BIOL LAB,BETHESDA,MD 20892
关键词
D O I
10.1021/bi00169a011
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Resonance Raman spectroscopy is used to monitor the redox state of heme a and heme a3 centers in cyanide-inhibited and native cytochrome oxidase during potentiometric titrations. Specific vibrational modes are resolved for each reduced heme with 441.6-nm excitation while oxidized species show vanishingly small Raman intensities. The voltage dependencies of the Raman intensities of reduced heme a and reduced heme a3 modes are quantitatively measured and used to extract heme a and a3 midpoint potentials. In the cyanide-bound enzyme, in which heme a3 remains in the oxidized state, the Raman data indicate that heme a centers exhibit complex Nernstian behavior with two E(m) values near 350 and 260 mV. In the native enzyme, this resonance Raman-potentiometric method reveals significantly different redox behavior for the two hemes. Heme a centers are described by two effective E(m) values near 350 and 220 mV, while heme a3 centers have lower E(m) values near 260 and 200 mV. Singular value decomposition analysis of optical spectral changes supports the Raman data. These results are in contrast to models of cytochrome oxidase redox behavior in which heme a and heme a3 are thought to have essentially identical midpoint potentials.
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页码:699 / 707
页数:9
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