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Clarke, Thomas A. 
ORCID: https://orcid.org/0000-0002-6234-1914, Kemp, Gemma L., Van Wonderen, Jessica H., Doyle, Rosie-Marie A. S., Cole, Jeffrey A., Tovell, Nick, Cheesman, Myles R., Butt, Julea N. ORCID: https://orcid.org/0000-0002-9624-5226, Richardson, David J. ORCID: https://orcid.org/0000-0002-6847-1832 and Hemmings, Andrew M. ORCID: https://orcid.org/0000-0003-3053-3134
(2008)
Role of a conserved gluatmine residue in tuning the catalytic activity of Escherichia coli cytochrome c nitrite reductase.
Biochemistry, 47 (12).
pp. 3789-3799.
Abstract
The pentaheme cytochrome c nitrite reductase (NrfA) of Escherichia coli is responsible for nitrite reduction during anaerobic respiration when nitrate is scarce. The NrfA active site consists of a hexacoordinate high-spin heme with a lysine ligand on the proximal side and water/hydroxide or substrate on the distal side. There are four further highly conserved active site residues including a glutamine (Q263) positioned 8 Å from the heme iron for which the side chain, unusually, coordinates a conserved, essential calcium ion. Mutation of this glutamine to the more usual calcium ligand, glutamate, results in an increase in the Km for nitrite by around 10-fold, while Vmax is unaltered. Protein film voltammetry showed that lower potentials were required to detect activity from NrfA Q263E when compared with native enzyme, consistent with the introduction of a negative charge into the vicinity of the active site heme. EPR and MCD spectroscopic studies revealed the high spin state of the active site to be preserved, indicating that a water/hydroxide molecule is still coordinated to the heme in the resting state of the enzyme. Comparison of the X-ray crystal structures of the as-prepared, oxidized native and mutant enzymes showed an increased bond distance between the active site heme Fe(III) iron and the distal ligand in the latter as well as changes to the structure and mobility of the active site water molecule network. These results suggest that an important function of the unusual Q263-calcium ion pair is to increase substrate affinity through its role in supporting a network of hydrogen bonded water molecules stabilizing the active site heme distal ligand.
| Item Type: | Article |
|---|---|
| Faculty \ School: | Faculty of Science > School of Biological Sciences Faculty of Science > School of Chemistry (former - to 2024) |
| UEA Research Groups: | Faculty of Science > Research Groups > Molecular Microbiology Faculty of Science > Research Groups > Biophysical Chemistry (former - to 2017) Faculty of Science > Research Groups > Organisms and the Environment Faculty of Science > Research Groups > Plant Sciences Faculty of Science > Research Groups > Chemistry of Light and Energy Faculty of Science > Research Groups > Chemistry of Life Processes Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry Faculty of Science > Research Groups > Energy Materials Laboratory |
| Depositing User: | EPrints Services |
| Date Deposited: | 01 Oct 2010 13:36 |
| Last Modified: | 24 Sep 2024 09:29 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/192 |
| DOI: | 10.1021/bi702175w |
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