The membrane subunit (NarI) of Escherichia coli nitrate reductase A (NarGHI) contains two btype hemes, both of which are the highly anisotropic low-spin type. Heme b D 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 b D 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 b D sensitive to Q-site occupancy. As such, we hypothesize that the heterogeneity in the heme b D 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 g z components of heme b D 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 b D exhibits an E m,8 of −35 mV and a pH dependence of −40 mV pH −1 . In the quinone-free state, however, heme b D titrates with an E m,8 of +25 mV and a pH dependence of −59 mV pH −1 . We hypothesize that quinone binding modulates the electrochemical properties of heme b D as well as its EPR properties.Nitrate reductase A (NarGHI) from Escherichia coli is a membrane-bound quinol:nitrate oxidoreductase that is expressed under anaerobic conditions in the presence of nitrate. 1 It The authors declare no competing financial interest. Supporting InformationResults of the cytochrome bd and bo 3 deletions on NarGHI EPR heme spectra as well as anaerobic growth curves for NarGHI, NarGHI-K86A, NarGHI-G65A, and the background strain, LCB79. This material is available free of charge via the Internet at http:// pubs.acs.org. CIHR Author Manuscript CIHR Author Manuscript CIHR Author Manuscriptfunctions as a terminal reductase, coupling quinol oxidation to nitrate reduction, and contributes to the generation of a proton electrochemical potential across the cytoplasmic membrane. 2 NarGHI comprises a catalytic subunit (NarG, 140 kDa), an electron-transfer subunit (NarH, 58 kDa), and a membrane anchor subunit (NarI, 26 kDa). NarG contains a molybdo-bis(pyranopterin guanine dinucleotide) (Mo-bisPGD) cofactor that is the site of nitrate reduction as well as a single tetranuclear iron-sulfur ([4Fe-4S]) cluster known as FS0. NarH contains three [4Fe-4S] clusters (FS1-FS3) and one trinuclear iron-sulfur cluster ([3Fe-4S], FS4). NarI anchors the NarGH subunits to the inside of the cytoplasmic membrane and contains two hemes b that are proximal (b P ) and distal (b D ) to the NarGH subunits, respectively. Overall, these subunits provide a molecular scaffold for an electrontransfer relay connecting the site of quinol oxidation adjacent to heme b D in NarI (the Qsite) with the...