van Dam P J, Reijerse E J, Hagen W R
Department of Molecular Spectroscopy, University of Nijmegen, The Netherlands.
Eur J Biochem. 1997 Sep 1;248(2):355-61. doi: 10.1111/j.1432-1033.1997.00355.x.
The active H-cluster of the Fe-hydrogenases from Megasphaera elsdenii and Desulfovibrio vulgaris (strain Hildenborough) has been investigated with one- and two-dimensional pulsed EPR spectroscopy. In both complexes the coordination of a nitrogen-containing ligand was found. The unusual quadrupole interaction parameters (D. vulgaris: quadrupole coupling constant, K = 1.20 MHz, asymmetry parameter eta = 0.32, M. elsdenii: K = 1.23 MHz, eta = 0.25) indicate a non-protein type of nitrogen and are consistent with cyanide as ligand to the H-cluster. The additional interactions measured on the EPR signal of the inactivated H-cluster in D. vulgaris hydrogenase are consistent with an imidazole interaction similar to that found in Rieske-type iron-sulfur clusters. Since a His residue near the putative H-cluster binding motif of Cys residues, His371, is the only conserved His in Fe-hydrogenases, it is a likely candidate for the base that accepts the proton in the heterolytic cleavage of molecular hydrogen. The inactivation of the enzyme is accompanied by direct binding of the imidazole ring to the H-cluster.
利用一维和二维脉冲电子顺磁共振波谱对来自埃氏巨球型菌和普通脱硫弧菌（希登伯勒菌株）的铁氢化酶的活性H簇进行了研究。在这两种复合物中均发现了含氮配体的配位情况。异常的四极相互作用参数（普通脱硫弧菌：四极耦合常数，K = 1.20 MHz，不对称参数η = 0.32；埃氏巨球型菌：K = 1.23 MHz，η = 0.25）表明存在一种非蛋白质类型的氮，并且与氰化物作为H簇的配体相符。在普通脱硫弧菌氢化酶失活的H簇的电子顺磁共振信号上测得的额外相互作用与在 Rieske 型铁硫簇中发现的咪唑相互作用相似。由于在假定的半胱氨酸残基H簇结合基序附近的一个组氨酸残基His371是铁氢化酶中唯一保守的组氨酸，它很可能是在分子氢异裂裂解中接受质子的碱基的候选者。酶的失活伴随着咪唑环与H簇的直接结合。
