3IR5

Crystal structure of NarGHI mutant NarG-H49C

3IR5 の概要

• エントリーDOI: 10.2210/pdb3ir5/pdb
• 関連するPDBエントリー: 1Q16 3IR6 3IR7
• 分子名称: Respiratory nitrate reductase 1 alpha chain, Respiratory nitrate reductase 1 beta chain, Respiratory nitrate reductase 1 gamma chain, ... (10 entities in total)
• 機能のキーワード: oxidoreductase, nitrate reductase, electron transfer, membrane protein, 4fe-4s, cell membrane, electron transport, iron, iron-sulfur, membrane, metal-binding, molybdenum, nitrate assimilation, transport, 3fe-4s, cell inner membrane, formylation, heme, transmembrane
• 由来する生物種: Escherichia coli K-12; 詳細
• 細胞内の位置: Cell membrane; Peripheral membrane protein: P09152 P11349; Cell inner membrane; Multi-pass membrane protein: P11350
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 229283.48

構造登録者

Bertero, M.G.,Rothery, R.A.,Weiner, J.H.,Strynadka, N.C.J. (登録日: 2009-08-21, 公開日: 2010-01-05, 最終更新日: 2024-11-06)

主引用文献

Rothery, R.A.,Bertero, M.G.,Spreter, T.,Bouromand, N.,Strynadka, N.C.,Weiner, J.H.
Protein crystallography reveals a role for the FS0 cluster of Escherichia coli nitrate reductase A (NarGHI) in enzyme maturation.
J.Biol.Chem., 285:8801-8807, 2010

PubMed Abstract: 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 Mo-bisPGD 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 approximately 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.

PubMed: 20053990
DOI: 10.1074/jbc.M109.066027

実験手法

X-RAY DIFFRACTION (2.3 Å)