Woodruff W H, Einarsdóttir O, Dyer R B, Bagley K A, Palmer G, Atherton S J, Goldbeck R A, Dawes T D, Kliger D S
INC-4, Los Alamos National Laboratory, NM 87545.
Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2588-92. doi: 10.1073/pnas.88.6.2588.
Time-resolved electronic absorption, infrared, resonance Raman, and magnetic circular dichroism spectroscopies are applied to characterization of the intermediate that is formed within 20 ps after photodissociation of CO from cytochrome a3 in reduced cytochrome oxidase. This intermediate decays with the same half-life (approximately 1 microseconds) as the post-photodissociation CU+B-CO species previously observed by time-resolved infrared. The transient UV/visible spectra, kinetics, infrared, and Raman evidence suggest that an endogenous ligand is transferred from CuB to Fea3 when CO binds to CuB, forming a cytochrome a3 species with axial ligation that differs from the reduced unliganded enzyme. The time-resolved magnetic circular dichroism results suggest that this transient is high-spin and, therefore, five-coordinate. Thus we infer that the ligand from CuB binds on the distal side of cytochrome a3 and displaces the proximal histidine imidazole. This remarkable mechanistic feature is an additional aspect of the previously proposed "ligand-shuttle" activity of the CuB/Fea3 pair. We speculate as to the identity of the ligand that is transferred between CuB and Fea3 and suggest that the ligand shuttle may play a functional role in redox-linked proton translocation by the enzyme.
时间分辨电子吸收光谱、红外光谱、共振拉曼光谱和磁圆二色光谱被用于表征还原型细胞色素氧化酶中细胞色素a3的CO光解离后20皮秒内形成的中间体。该中间体的衰变半衰期（约1微秒）与先前通过时间分辨红外观察到的光解离后CU+B-CO物种相同。瞬态紫外/可见光谱、动力学、红外和拉曼证据表明，当CO与CuB结合时，一个内源性配体从CuB转移到Fea3，形成一种细胞色素a3物种，其轴向配体与还原的未配位酶不同。时间分辨磁圆二色性结果表明，这种瞬态是高自旋的，因此是五配位的。因此我们推断，来自CuB的配体结合在细胞色素a3的远端，并取代近端组氨酸咪唑。这一显著的机制特征是先前提出的CuB/Fea3对“配体穿梭”活性的一个额外方面。我们推测了在CuB和Fea3之间转移的配体的身份，并表明配体穿梭可能在该酶的氧化还原相关质子转运中发挥功能作用。
