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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:Stoicheometries and rates of proton translocation associated with respiratory reduction of NO3- have been measured for spheroplasts of Escherichia coli grown anaerobically in the presence of NO3-. Observed stoicheiometries [leads to H+/NO3- ratio; P. Mitchell (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin] were approx. 4 for L-malate oxidation and approx. 2 for succinate, D-lactate and glycerol oxidation. Measurements of the leads to H+/2e- ratio with formate as the reductant and oxygen or NO3- as the oxidant were complicated by pH changes associated with formate uptake and CO2 formation. Nevertheless, it was possible to conclude that the site of formate oxidation is on the inner aspect of the cytoplasmic membrane, that the leads to H+/O ratio for formate oxidation is approx. 4, and that the leads to H+/NO3- ratio is greater than 2. Measurements of the rate of NO3- penetration into osmotically sensitive spheroplasts demonstrated an electrogenic entry of NO3- anion. The permeability coefficient for nitrate entry at 30 degrees C was between 10(-9) and 10(-10) cm- s(-1). The calculated rate of nitrate entry at the concentration typically used for the assay of nitrate reductase (EC 1.7.99.4) activity was about 0.1% of that required to support the observed rate of nitrate reduction by reduced Benzyl Viologen. Measurements of the distribution of nitrate between the intracellular and extracellular spaces of a haem-less mutant, de-repressed for nitrate reductase but unable to reduce nitrate by the respiratory chain, showed that, irrespective of the presence or the absence of added glucose, nitrate was not concentrated intracellularly. Osmotic-swelling experiments showed that the rate of diffusion of azid anion across the cytoplasmic membrane is relatively low in comparison with the fast diffusion of hydrazoic acid. The inhibitory effect of azide on nitrate reductase was not altered by treatments that modify pH gradients across the cytoplasmic membrane. It is concluded that the nitrate-reducing azide-sensitive site of nitrate reductase is located on the outer aspect of the cytoplasmic membrane. The consequences of this location for mechanisms of proton translocation driven by nitrate reduction are discussed, and lead to the proposal that the nitrate reductase of the cytoplasmic membrane is vectorial, reducing nitrate on the outer aspect of the membrane with 2H+ and 2e- that have crossed from the inner aspect of the membrane.

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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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