Role of a Conserved Glutamine Residue in Tuning the Catalytic Activity of Escherichia coli Cytochrome c Nitrite Reductase
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 A 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 K m for nitrite by around 10-fold, while V max 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.
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NrfA contains five CXXC motifs for covalent heme attachment.
“NrfA contains five CXXC motifs for covalent heme attachment59.”
From AF2Complex predicts direct physical interactions in multimeric proteins with deep learning (2022) · cites this paper as [59]
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- Authors
- Clarke, T. A., Kemp, G. L., Wonderen, J. H. V., Doyle, R.-M. A. S., Cole, J. A., Tovell, N., Cheesman, M. R., Butt, J. N., Richardson, D. J., & Hemmings, A. M.
- Affiliations
- University of East Anglia (GB)
- Corresponding author
- Richardson, David J.
- Hemmings, Andrew M.
- ORCID
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2008-03-01
- Volume
- 47
- Issue
- 12
- Pages
- 3789-3799
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W1967590221
- Cited by
- 40
- References
- 39
- Citations by year
- 2025: 4 · 2024: 1 · 2022: 3 · 2021: 3 · 2020: 2 · 2019: 1 · 2018: 4 · 2016: 1 · 2015: 3 · 2014: 2 · 2013: 3 · 2012: 2
- FWCI
- 2.22
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- University of Birmingham Research Portal (University of Birmingham) (submittedVersion)
- UEA Digital Repository (University of East Anglia) (submittedVersion)
- Funders
- Wellcome Trust (0162178)
- Biotechnology and Biological Sciences Research Council (B18695)
- Topics
- Physical Sciences › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
- Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Metalloenzymes and iron-sulfur proteins
- Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
- Keywords
- cytochrome c nitrite reductase, nrfA, calcium coordination, substrate affinity, nitrite reduction, high-spin heme, Escherichia coli
- MeSH terms
- Binding Sites, Catalysis, Circular Dichroism, Crystallization, Cytochrome c Group, Electron Spin Resonance Spectroscopy, Escherichia coli, Glutamine, Models, Molecular, Crystallography, X-Ray, Glutamic Acid, Amino Acid Substitution
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