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

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.

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