Catalytic Protein Film Voltammetry from a Respiratory Nitrate Reductase Provides Evidence for Complex Electrochemical Modulation of Enzyme Activity
Abstract
The first step in the respiratory reduction of nitrate to dinitrogen in Paracoccus pantotrophus is catalyzed by the quinol-nitrate oxidoreductase NarGHI. This membrane-anchored protein directs electrons from quinol oxidation at the membrane anchor, NarI, to the site of nitrate reduction in the membrane extrinsic [Fe-S] cluster and Mo-bis-MGD containing dimer, NarGH. Liberated from the membrane, NarGH retains its nitrate reductase activity and forms films on graphite and gold electrodes within which direct and facile exchange of electrons between the electrode and the enzyme occurs. Protein film voltammetry has been used to define the catalytic behavior of NarGH in the potential domain and a complex pattern of reversible, nitrate concentration dependent modulation of activity has been resolved. At low nitrate concentrations the local maximum observed in the catalytic current-potential profile reveals how NarGH can catalyze nitrate reduction via two pathways having distinct specificity constants, k(obs)(cat)/K(obs)(M). Catalysis is directed to occur via one of the pathways by an electrochemical event within NarGH. On increasing the nitrate concentration, the local maximum in the catalytic current becomes less distinct, and the catalytic waveform adopts an increasingly sigmoidal form. A pattern of voltammetry similar to that observed during nitrate reduction is observed during reduction of the stereochemically distinct substrate chlorate. Centers whose change of oxidation state may define the novel catalytic voltammetry of NarGH have been identified by EPR-monitored potentiometric titrations and mechanisms by which the electrochemistry of Mo-bis-MGD or [Fe-S] clusters can account for the observed behavior are discussed.
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PFV experiments on Nap and Nar enzymes reveal different kinetic parameters.
“PFV experiments on Nap and Nar enzymes reveal different kinetic parameters.217–220”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [217]
For the NarGHI complex, there are two states capable of substrate reduction.
“for the NarGHI complex, there are two states capable of substrate reduction”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [217]
These two states are coupled by a redox reaction.
“these two states are coupled by a redox reaction.217”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [217]
In the NarGHI complex, the Mo(v) state associates more rapidly with nitrate than the Mo(iv) state.
“the Mo(v) state associates more rapidly with nitrate than the Mo(iv) state”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [217]
The Mo(v):NO3 complex is more stable than the Mo(iv):NO3 complex.
“the Mo(v):NO3 complex is more stable.”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [217]
At higher substrate concentrations where catalysis becomes enzyme-limited, the pathway involving the Mo(v) state, which is also faster, becomes more involved.
“at higher substrate concentrations where the catalysis becomes enzyme-limited, the pathway involving the Mo(v) state, which is also faster, becomes more involved.”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [217]
In this scenario, Mo(v) is reduced to Mo(iv) in a substrate bound form.
“In this scenario, Mo(v) is reduced to Mo(iv) in a substrate bound form.”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [217]
At lower nitrate concentrations the substrate is transformed effectively via a slower Mo(iv) pathway.
“At lower nitrate concentrations the substrate is transformed effectively via a slower Mo(iv) pathway.”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [217]
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Self-citation
- Authors
- Anderson, L. J., Richardson, D. J., & Butt, J. N.
- Affiliations
- University of East Anglia (GB)
- Corresponding author
- Butt, Julea N.
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2001-08-25
- Volume
- 40
- Issue
- 38
- Pages
- 11294-11307
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W2027157827
- Cited by
- 113
- References
- 20
- Citations by year
- 2026: 1 · 2025: 4 · 2024: 1 · 2023: 2 · 2022: 3 · 2021: 3 · 2020: 6 · 2019: 3 · 2018: 3 · 2017: 4 · 2016: 6 · 2015: 3 · 2014: 5 · 2013: 7 · 2012: 3
- FWCI
- 7.37
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- UEA Digital Repository (University of East Anglia) (submittedVersion)
- Topics
- Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Metalloenzymes and iron-sulfur proteins
- Physical Sciences › Engineering › Electrical and Electronic Engineering › Electrochemical sensors and biosensors
- Physical Sciences › Environmental Science › Environmental Engineering › Microbial Fuel Cells and Bioremediation
- Keywords
- respiratory nitrate reductase, Paracoccus pantotrophus, protein film voltammetry, nitrate reduction, chlorate reduction, iron-sulfur clusters, enzyme kinetics, substrate specificity, electrochemical titration
- MeSH terms
- Catalysis, Cell Membrane, Electrochemistry, Electron Spin Resonance Spectroscopy, Hydrogen-Ion Concentration, Hydroquinones, Kinetics, Models, Chemical, Models, Theoretical, Nitrate Reductases, Nitrates, Oxygen Consumption, Paracoccus, Nitrate Reductase
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