3IR5: Crystal structure of NarGHI mutant NarG-H49C

Citation:
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.
PDB ID: 3IR5Download
MMDB ID: 155896
PDB Deposition Date: 2009/8/21
Updated in MMDB: 2017/11
Experimental Method:
x-ray diffraction
Resolution: 2.3  Å
Source Organism:
Similar Structures:
Biological Unit for 3IR5: trimeric; determined by author and by software (PISA)
Molecular Components in 3IR5
Label Count Molecule
Proteins (3 molecules)
1
Respiratory Nitrate Reductase 1 Alpha Chain(Gene symbol: narG)
Molecule annotation
1
Respiratory Nitrate Reductase 1 Beta Chain(Gene symbol: narH)
Molecule annotation
1
Respiratory Nitrate Reductase 1 Gamma Chain(Gene symbol: narI)
Molecule annotation
Chemicals (11 molecules)
1
2
2
1
3
4
4
1
5
1
6
2
* Click molecule labels to explore molecular sequence information.

Citing MMDB
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