Hofrichter J, Henry E R, Sommer J H, Deutsch R, Ikeda-Saito M, Yonetani T, Eaton W A
Biochemistry. 1985 May 21;24(11):2667-79. doi: 10.1021/bi00332a012.
Hybrid hemoglobins were prepared in which cobalt was substituted for the heme iron in either the alpha or beta subunits. Transient optical absorption spectra were measured at room temperature for these hybrids at time intervals between 0 and 50 ms following photodissociation of the carbon monoxide complex with 10-ns laser pulses. The cobalt porphyrins do not bind carbon monoxide, making it possible to investigate the time-resolved response of the cobalt-containing subunits to photodissociation of carbon monoxide in the iron-containing subunits. At the same time the response of the iron-containing subunits to the photolysis event can be studied, permitting an independent determination of the kinetics of ligand rebinding and conformational changes in the alpha and beta subunits of an intact tetramer. The data were analyzed by using singular-value decomposition to obtain the kinetic progress curve for ligand rebinding, the deoxyheme and cobalt porphyrin spectral changes, and the time course of these spectral changes. The geminate rebinding kinetics following photodissociation of alpha(Co)2 beta(Fe-CO)2 were very similar to those found unsubstituted hemoglobin, alpha(Fe-CO)2 beta(Fe-CO)2, indicating equivalence of the geminate kinetics for alpha and beta subunits within the R-state tetramer. The results for alpha(Fe-CO)2 beta(Co)2 were consistent with this conclusion, even though the analysis was complicated by the presence of comparable populations of R- and T-state species. Comparison of the deoxyheme spectral changes and relaxation times among the three molecules indicated that both alpha and beta subunits contribute to the deoxyheme spectral changes that signal tertiary and quaternary conformational changes in the unsubstituted tetramer. The response of the cobalt porphyrins to photodissociation was similar in the two hybrids. No structural changes were detected in the cobalt-containing subunits until the second tertiary conformational change in the iron-containing subunits observed at 1-2 microseconds. Much larger structural changes, as judged by the amplitude of the spectral changes, occurred in the cobalt-containing subunits concomitant with the R----T quaternary change at about 20 microseconds.
制备了杂合血红蛋白，其中钴取代了α或β亚基中的血红素铁。在室温下，用10纳秒激光脉冲使一氧化碳复合物光解离后，在0至50毫秒的时间间隔内测量这些杂合体的瞬态光吸收光谱。钴卟啉不结合一氧化碳，这使得研究含钴亚基对含铁亚基中一氧化碳光解离的时间分辨响应成为可能。同时，可以研究含铁亚基对光解事件的响应，从而独立测定完整四聚体α和β亚基中配体重新结合和构象变化的动力学。通过使用奇异值分解分析数据，以获得配体重新结合的动力学进程曲线、脱氧血红素和钴卟啉的光谱变化以及这些光谱变化的时间进程。α(Co)2β(Fe-CO)2光解离后的双分子重新结合动力学与未取代的血红蛋白α(Fe-CO)2β(Fe-CO)2非常相似，表明R态四聚体内α和β亚基的双分子动力学相当。α(Fe-CO)2β(Co)2的结果与这一结论一致，尽管由于存在相当数量的R态和T态物种，分析变得复杂。对这三种分子中脱氧血红素光谱变化和弛豫时间的比较表明，α和β亚基都对未取代四聚体中指示三级和四级构象变化的脱氧血红素光谱变化有贡献。两种杂合体中钴卟啉对光解离的响应相似。在含铁亚基在1-2微秒时观察到的第二次三级构象变化之前，未检测到含钴亚基的结构变化。根据光谱变化的幅度判断，在约20微秒时，含钴亚基伴随着R→T四级变化发生了更大的结构变化。
