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Q-site occupancy defines heme heterogeneity in Escherichia coli nitrate reductase A (NarGHI).

ABSTRACT: The membrane subunit (NarI) of Escherichia coli nitrate reductase A (NarGHI) contains two b-type hemes, both of which are the highly anisotropic low-spin type. Heme bD is distal to NarGH and constitutes part of the quinone binding and oxidation site (Q-site) through the axially coordinating histidine-66 residue and one of the heme bD propionate groups. Bound quinone participates in hydrogen bonds with both the imidazole of His66 and the heme propionate, rendering the EPR spectrum of the heme bD sensitive to Q-site occupancy. As such, we hypothesize that the heterogeneity in the heme bD EPR signal arises from the differential occupancy of the Q-site. In agreement with this, the heterogeneity is dependent upon growth conditions but is still apparent when NarGHI is expressed in a strain lacking cardiolipin. Furthermore, this heterogeneity is sensitive to Q-site variants, NarI-G65A and NarI-K86A, and is collapsible by the binding of inhibitors. We found that the two main gz components of heme bD exhibit differences in reduction potential and pH dependence, which we posit is due to differential Q-site occupancy. Specifically, in a quinone-bound state, heme bD exhibits an Em,8 of -35 mV and a pH dependence of -40 mV pH(-1). In the quinone-free state, however, heme bD titrates with an Em,8 of +25 mV and a pH dependence of -59 mV pH(-1). We hypothesize that quinone binding modulates the electrochemical properties of heme bD as well as its EPR properties.

SUBMITTER: Fedor JG

PROVIDER: S-EPMC4059744 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Q-site occupancy defines heme heterogeneity in Escherichia coli nitrate reductase A (NarGHI).

Fedor Justin G JG Rothery Richard A RA Giraldi Karissa S KS Weiner Joel H JH

Biochemistry 20140312 11

The membrane subunit (NarI) of Escherichia coli nitrate reductase A (NarGHI) contains two b-type hemes, both of which are the highly anisotropic low-spin type. Heme bD is distal to NarGH and constitutes part of the quinone binding and oxidation site (Q-site) through the axially coordinating histidine-66 residue and one of the heme bD propionate groups. Bound quinone participates in hydrogen bonds with both the imidazole of His66 and the heme propionate, rendering the EPR spectrum of the heme bD ...[more]

PMID: 24592999

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Project description:Cellular respiration is a fundamental process undergone within energy-transducing membranes through the redox activity of multimeric protein assemblies, so-called respiratory complexes. In most cases, electron transport is ensured by lipophilic molecules, quinones, serving as electron shuttles. This redox activity is coupled to the net translocation of protons across the membrane thereby establishing an electrochemical gradient, the protonmotive force (pmf). Such a gradient powers the transport of molecules (such as proteins, ions or antibiotics) and ATP synthesis but was also shown to participate in protein localization in prokaryotes. Importantly, energy-transducing membranes show a high level of organization with heterogeneous distribution of respiratory complexes as observed across several bacterial lineages. Although electron transport in energy-transducing membranes is considered to be a kinetic process coupled to the diffusion of all reactants and in particular quinones, it is not clear to which extent subcellular distribution of respiratory complexes can have an influence on the overall kinetics. We previously evidenced polar clustering of nitrate reductase, NarGHI, an anaerobic respiratory complex in the gut bacterium Escherichia coli. Such spatial organization was shown to directly impact the electron flux of the associated respiratory chain. While dynamic localization of such complex plays an important role in controlling respiration, the mechanism by which it impacts the electron flux is not understood. Yet, under such conditions where nitrate is the sole terminal electron acceptor, functioning of the electron transport chain in enteric bacteria such as E. coli and Salmonella typhimurium is associated with the production of nitric oxide (NO) mainly resulting from the reduction of nitrite by nitrate reductase. As a consequence, both E. coli and S. typhimurium produce a multitude of enzymes controlling NO homeostasis, the primary source of nitrosative stress. Protein S-nitrosylation is a ubiquitous NO-dependent post-translational modification of cysteine that regulates protein structure and function. Recently, Seth et al demonstrated that, in absence of oxygen, NO oxidation is mainly catalyzed by the hybrid cluster protein Hcp allowing its further reaction with thiols of a broad spectrum of proteins. Among Hcp-dependent S-nitrosylated targets are the nitrate reductase complex, multiple metabolic enzymes and the transcription factor OxyR whose S-nitrosylation entails a distinct nitrosative stress regulon. Optimizing electron transport through clustering of nitrate reductase will lead to nitrite accumulation which may in turn be responsible for NO production. Here, the electron-donating respiratory complex, the formate dehydrogenase, FdnGHI was shown to cluster at the poles under nitrate respiring conditions. Its proximity with the nitrate reductase complex was confirmed by evaluating the interactome of the later under distinct metabolic conditions reported to impact its subcellular organization. All the identified partners were exclusively found when cells were grown under nitrate respiration. The proximity of two respiratory complexes provides mechanistic explanation on the importance of subcellular organization onto quinone pool turnover. Noteworthy is the identification of several components playing key roles in the protection against endogenous nitrosative stress.

Dataset Information

Q-site occupancy defines heme heterogeneity in Escherichia coli nitrate reductase A (NarGHI).

Publications

Q-site occupancy defines heme heterogeneity in Escherichia coli nitrate reductase A (NarGHI).

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