固氮酶活性中心催化N2和H+还原位点的分析

Site-directed mutagenesis and gene-replacement procedures were used to construct mutant nitrogenase of klebsiella pneumoniae. The α-subunit residue 194 histidine of polypeptide environment of FeMo-cofactor was substituted by glutamine 。The phenotype of the mutant strain is Nif-,but in vivo,its acetylene reduction activity is 33% of that of wild type. Camparisions of catalytic properties of the C2H2、 N2 and H+ reduction of the altered nitrogenase and the wild one show that at the Fe/MoFe molar ratio of ca. 11, Kpα194Gln nitrogenase reach its highest acetylene reduction activity that is 75% of wild type. Similar to Azotobacter vinelandii a195Gln mutant nitrogenase, Kpα194Gln nitrogenase has the weak nitrogen fixation ability which is about 1.5% of the wild type, but the total elctronic flux is also up to 65% of wild type,which is different from Av Gln a195 mutant nitrogenase. those evidence suggests that (1)α-subunit residue 194 histidine be indispensable to the dinitrogen reduction and alteration at this site effects efficiency of elctronic transfer markedly; (2)the NH-S hydrogen bond between His194 and S2B may serve as the main channel of elctronic transfer to FeMoco and the six Fe atoms ligand to the three central S will have a role involved in the dinitrogen reduction;(3) there shoud have an H2 evolution site indepented on the N2 binding and reduction. At the same time, chemical simulation of biological nitrogen fixation research was performed and the activity of the simulated K[Fe4S3(NO)7] catalyzer was detected.

通过对野生型及突变型棕色固氮菌（Azotobacter vinelandii）固氮酶在不同条件下，催化N2还原和H+还原活性的对比研究，分析确定N2和H+在固氮酶活性中心原子簇上的络合和还原位点及其电子通道，为化学模拟生物固氮提供理论依据。