期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 285, 期 12, 页码 8801-8807出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M109.066027
关键词
-
资金
- National Institutes of Health [GMO68451-01]
- Canadian Institutes of Health Research [MOP15292]
- Canada Foundation for Innovation
- Alberta Heritage Foundation for Medical Research
We have used site-directed mutagenesis, EPR spectroscopy, redox potentiometry, and protein crystallography to monitor assembly of the FS0 [4Fe-4S] cluster and molybdo-bis(pyranopterin guanine dinucleotide) cofactor (Mo-bisPGD) of the Escherichia coli nitrate reductase A (NarGHI) catalytic subunit (NarG). Cys and Ser mutants of NarG-His(49) both lack catalytic activity, with only the former assembling FS0 and Mo-bisPGD. Importantly, both prosthetic groups are absent in the NarG-H49S mutant. EPR spectroscopy of the Cys mutant reveals that the E-m value of the FS0 cluster is decreased by at least 500 mV, preventing its participation in electron transfer to the MobisPGD cofactor. To demonstrate that decreasing the FS0 cluster E-m results in decreased enzyme activity, we mutated a critical Arg residue (NarG-Arg(94)) in the vicinity of FS0 to a Ser residue. In this case, the E-m of FS0 is decreased by 115 mV, with a concomitant decrease in enzyme turnover to similar to 30% of the wild type. Analysis of the structure of the NarG-H49S mutant reveals two important aspects of NarGHI maturation: (i) apomolybdo-NarGHI is able to bind GDP moieties at their respective P and Q sites in the absence of the Mo-bisPGD cofactor, and (ii) a critical segment of residues in NarG, (49)HGVNCTG(55), must be correctly positioned to ensure holoenzyme maturation.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据