Rousseau D L, Singh S, Ching Y C, Sassaroli M
AT&T Bell Laboratories, Murray Hill, New Jersey 07974.
J Biol Chem. 1988 Apr 25;263(12):5681-5.
We report the first resonance Raman scattering studies of NO-bound cytochrome c oxidase. Resonance Raman scattering and optical absorption spectra have been obtained on the fully reduced enzyme (a2+, a2+(3) NO) and the mixed valence enzyme (a3+, a2+(3) NO). Clear vibrational frequency shifts are detected in the lines associated with cytochrome a in comparing the two redox states. With 441.6 nm excitation the fully reduced preparation yields a spectrum similar to that of carbon monoxide-bound cytochrome c oxidase and is dominated by the spectrum of reduced cytochrome a. In contrast, in the mixed valence preparation no contributions from reduced cytochrome a are evident in the spectrum, verifying that this heme is no longer in the Fe2+ state. In the mixed valence NO-bound samples, a line appears at approximately 545 cm-1, a frequency similar to that found in NO-bound hemoglobin and myoglobin and assigned as an Fe-N-O-bending mode in those proteins. We do not detect this line in the spectrum of the fully reduced NO-bound enzyme. The carbonyl line of the cytochrome a3 heme formyl group in the fully reduced NO-bound enzyme appears at approximately equal to 1666 cm-1 in the resonance Raman spectrum. In the mixed valence NO-bound preparation the frequency of the carbonyl line increases by 1.2 cm-1 to approximately equal to 1667 cm-1. Thus, modes in cytochrome a2+(3) NO are sensitive to the redox state of the cytochrome a and/or CuA centers. We propose that the redox sensitivity of the formyl mode and the Fe-N-O mode results from an interaction between cytochrome a2+(3) (NO) and the cytochrome a-CuA pair, and is linked to the cytochrome a3 (NO) by the coupling between CuB and the NO-bound cytochrome a3 heme.
我们报道了首例关于一氧化氮结合型细胞色素c氧化酶的共振拉曼散射研究。已获得了完全还原态酶（a2+，a2+(3)NO）和混合价态酶（a3+，a2+(3)NO）的共振拉曼散射光谱和光吸收光谱。在比较这两种氧化还原状态时，在与细胞色素a相关的谱线中检测到了明显的振动频率位移。用441.6nm激发光时，完全还原态的样品产生的光谱与一氧化碳结合型细胞色素c氧化酶的光谱相似，且以还原态细胞色素a的光谱为主。相比之下，在混合价态样品的光谱中，还原态细胞色素a没有明显贡献，这证实该血红素不再处于Fe2+状态。在混合价态一氧化氮结合型样品中，一条谱线出现在约545cm-1处，该频率与一氧化氮结合型血红蛋白和肌红蛋白中的频率相似，在那些蛋白质中被指定为Fe-N-O弯曲模式。我们在完全还原态一氧化氮结合型酶的光谱中未检测到这条谱线。在共振拉曼光谱中，完全还原态一氧化氮结合型酶中细胞色素a3血红素甲酰基的羰基谱线出现在约1666cm-1处。在混合价态一氧化氮结合型样品中，羰基谱线的频率增加了1.2cm-1，约为1667cm-1。因此，细胞色素a2+(3)NO中的模式对细胞色素a和/或CuA中心的氧化还原状态敏感。我们提出，甲酰基模式和Fe-N-O模式的氧化还原敏感性源于细胞色素a2+(3)(NO)与细胞色素a-CuA对之间的相互作用，并通过CuB与一氧化氮结合型细胞色素a3血红素之间的耦合与细胞色素a3(NO)相连。
