Rothery R A, Magalon A, Giordano G, Guigliarelli B, Blasco F, Weiner J H
Medical Research Council Group in the Molecular Biology of Membranes, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
J Biol Chem. 1998 Mar 27;273(13):7462-9. doi: 10.1074/jbc.273.13.7462.
We have studied the effect of a mobAB mutation and tungstate on molybdo-molybdopterin-guanine dinucleotide (Mo-MGD) insertion into Escherichia coli nitrate reductase (NarGHI). Preparation of fluorescent oxidized derivatives of MGD (Form A and Form B) indicates that in a mobAB mutant there is essentially no detectable cofactor present in either the membrane-bound (NarGHI) or purified soluble (NarGH) forms of the enzyme. Electron paramagnetic resonance characterization of membrane-bound cofactor-deficient NarGHI suggests that it has altered electrochemistry with respect to the dithionite reducibility of the [Fe-S] clusters of NarH. Potentiometric titrations of membrane-bound NarGHI indicate that the NarH [Fe-S] clusters have midpoint potentials at pH 8.0 (Em,8.0 values) of +180 mV ([3Fe-4S] cluster), +130, -55, and -420 mV ([4Fe-4S] clusters) in a wild-type background and +180, +80, -35, and -420 mV in a mobAB mutant background. These data support the following conclusions: (i) a model for Mo-MGD biosynthesis and assembly into NarGHI in which both metal chelation and nucleotide addition to molybdopterin precede cofactor insertion; and (ii) the absence of Mo-MGD significantly affects Em,8.0 of the highest potential [4Fe-4S] cluster.
我们研究了mobAB突变和钨酸盐对钼-钼蝶呤-鸟嘌呤二核苷酸（Mo-MGD）插入大肠杆菌硝酸还原酶（NarGHI）的影响。MGD荧光氧化衍生物（A形式和B形式）的制备表明，在mobAB突变体中，无论是膜结合形式（NarGHI）还是纯化的可溶性形式（NarGH）的酶中，基本上都检测不到辅因子。膜结合的辅因子缺陷型NarGHI的电子顺磁共振表征表明，就NarH的[Fe-S]簇的连二亚硫酸盐还原性而言，其电化学性质发生了改变。膜结合的NarGHI的电位滴定表明，在野生型背景下，NarH的[Fe-S]簇在pH 8.0时的中点电位（Em,8.0值）为+180 mV（[3Fe-4S]簇）、+130、-55和-420 mV（[4Fe-4S]簇），而在mobAB突变体背景下为+180、+80、-35和-420 mV。这些数据支持以下结论：（i）Mo-MGD生物合成并组装到NarGHI中的模型，其中金属螯合和向钼蝶呤添加核苷酸都先于辅因子插入；（ii）Mo-MGD的缺失显著影响最高电位的[4Fe-4S]簇的Em,8.0。
