van Belzen R, de Jong A M, Albracht S P
E. C. Slater Institute for Biochemical Research, University of Amsterdam, The Netherlands.
Eur J Biochem. 1992 Nov 1;209(3):1019-22. doi: 10.1111/j.1432-1033.1992.tb17377.x.
The concentration of the iron-sulphur (Fe-S) cluster 1b, present in complex I or soluble high-molecular-mass NADH dehydrogenase, was determined using different methods. It was found that direct double integration of the EPR signal at temperatures higher than 40 K, as is commonly used in this field of research, results in a considerable overestimation of the concentration of cluster 1b. It is demonstrated that this is caused by contributions from the relaxation-broadened signals of the Fe-S clusters 2-4 in the enzyme. The correct way for determining the intensity of the EPR signal of cluster 1b is by comparison with a simulated line shape. It is concluded that the concentration of cluster 1b is half that of cluster 2. This corroborates our proposal based on presteady-state kinetic and inhibitor-titration studies [Van Belzen, R., Van Gaalen, M. C. M., Cuypers, P. A. & Albracht S. P. J. (1990) Biochim. Biophys Acta 1017, 152-159] that the minimal functional unit of mitochondrial NADH:ubiquinone oxidoreductase must be a heterodimer.
采用不同方法测定了存在于复合物I或可溶性高分子量NADH脱氢酶中的铁硫（Fe-S）簇1b的浓度。结果发现，在高于40K的温度下对EPR信号进行直接双积分（这是该研究领域常用的方法），会导致对簇1b浓度的显著高估。结果表明，这是由酶中铁硫簇2 - 4的弛豫加宽信号的贡献所致。确定簇1b的EPR信号强度的正确方法是与模拟线形进行比较。得出的结论是，簇1b的浓度是簇2的一半。这证实了我们基于预稳态动力学和抑制剂滴定研究[Van Belzen, R., Van Gaalen, M. C. M., Cuypers, P. A. & Albracht S. P. J. (1990) Biochim. Biophys Acta 1017, 152 - 159]提出的观点，即线粒体NADH：泛醌氧化还原酶的最小功能单位必定是异二聚体。
