Goldbeck R A, Einarsdóttir O, Dawes T D, O'Connor D B, Surerus K K, Fee J A, Kliger D S
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064.
Biochemistry. 1992 Oct 6;31(39):9376-87. doi: 10.1021/bi00154a008.
Near-UV-vis magnetic and natural circular dichroism (MCD and CD) spectra of oxidized, reduced, and carbonmonoxy-complexed cytochrome ba3, a terminal oxidase from the bacterium Thermus thermophilus, and nanosecond time-resolved MCD (TRMCD) and CD (TRCD) spectra of the unligated species formed after photodissociation of the CO complex are presented. The spectral contributions of individual cytochromes b and a3 to the Soret region MCD are identified. TRMCD spectroscopy is used to follow the spin state change (S = 0 to S = 2) in cytochrome a3(2+) following photodissociation of the CO complex. There is prompt appearance of the high-spin state after photolysis, as found previously in mammalian cytochrome oxidase [Goldbeck, R. A., Dawes, T. D., Einarsdóttir, O., Woodruff, W. H., & Kliger, D. S. (1991) Biophys. J. 60, 125-134]. Peak shifts of 1-10 nm appear in the TRMCD, TRCD, and time-resolved UV-vis absorption spectra of the photolyzed enzyme throughout its observable lifetime, indicating that the photolyzed enzyme does not relax to its equilibrium deliganded form before recombination with CO occurs hundreds of milliseconds later. Direct heme-heme interaction is not found in cytochrome ba3, but red-shifts in the MCD and absorption spectra of both cytochromes b and (photolyzed) a3 are correlated with a CO-liganded form of the protein. The long time (tau approximately greater than 1 s) needed for relaxation of the cytochrome b and a3 peaks to their static positions suggests that CO binding to a3 induces a global conformational change in the protein that weakly perturbs the MCD and absorption spectra of b and photolyzed a3. Fea3 binds CO more weakly in cytochrome ba3 than in cytochrome aa3. The MCD spectrum of reduced enzyme solution placed under 1 atm of CO contains a peak at 446 nm that shows approximately 30% of total cytochrome a3 remains pentacoordinate, high-spin.
本文展示了嗜热栖热菌末端氧化酶细胞色素ba3氧化态、还原态和一氧化碳复合态的近紫外可见磁圆二色光谱（MCD）和天然圆二色光谱（CD），以及CO复合物光解离后形成的未配位物种的纳秒时间分辨MCD（TRMCD）和CD（TRCD）光谱。确定了单个细胞色素b和a3对Soret区域MCD的光谱贡献。利用TRMCD光谱跟踪CO复合物光解离后细胞色素a3(2+)中的自旋态变化（S = 0至S = 2）。光解后迅速出现高自旋态，这与之前在哺乳动物细胞色素氧化酶中发现的情况一致[戈德贝克，R. A.，道斯，T. D.，埃纳尔斯多蒂尔，O.，伍德拉夫，W. H.，& 克利格，D. S.（1991年）《生物物理杂志》60卷，125 - 134页]。在整个可观测寿命期间，光解酶的TRMCD、TRCD和时间分辨紫外可见吸收光谱中出现了1 - 10纳米的峰位移，这表明光解酶在数百毫秒后与CO重新结合之前，不会松弛到其平衡的未配位形式。在细胞色素ba3中未发现直接的血红素 - 血红素相互作用，但细胞色素b和（光解后的）a3的MCD和吸收光谱中的红移与蛋白质的CO配位形式相关。细胞色素b和a3峰松弛到其静态位置所需的长时间（τ约大于1秒）表明，CO与a3的结合诱导了蛋白质的全局构象变化，这对b和光解后的a3的MCD和吸收光谱产生了微弱的扰动。在细胞色素ba3中，Fea3与CO的结合比在细胞色素aa3中更弱。置于1个大气压CO下的还原酶溶液的MCD光谱在446纳米处有一个峰，表明约30%的总细胞色素a3保持五配位、高自旋状态。
