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 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...

Demethylmenaquinol is a substrate of Escherichia coli nitrate reductase A (NarGHI) and forms a stable semiquinone intermediate at the NarGHI quinol oxidation site

“…Hemes b D and b P titrate as a single component that fits to n = 1 Nernstian curve with midpoint potential E′ m,7.5~− 10 mV and +50 mV, respectively (Fig. 5), in consistency with previously published data [37,40]. Importantly, the EPR spectrum of NarGHI-bound DMSK is not affected by the redox state of the two hemes.…”

“…Baseline drift is due to the presence of adventitious Fe 3+ giving an isotropic signal at g = 4.3. The minor peak at g~2.97 is associated to the presence of cytochrome bo 3 ubiquinol oxidase [37].…”

A New Paradigm for Electron Transfer through Escherichia coli Nitrate Reductase A

“…Heme b D often manifests as having two subpopulations with g z values of ∼3.35 and ∼3.2, which we propose are due to differential Q-site occupancies, whereas others have suggested the subpopulations arise from differential cardiolipin occupancies . Under anaerobic or microaerobic conditions, the g z feature of the heme b D spectrum manifests as a single peak at g = 3.35 (Figure ). ,, When present, these two subpopulations do exhibit different apparent reduction potentials, and thus, simple signal integration is not feasible . However, the two subpopulations collapse into one signal, albeit with a shifted g z , upon inhibitor binding.…”

“…However, before their behavior in the potential domain can be thoroughly analyzed, the homogeneity of these signals needs to be established. The g = 3.75 peak of heme b D is clearly resolved from signals of other moieties that might impact our analyses (Figure ), as there are no signals in this region in membranes lacking NarGHI. ,,, FS4 is more complicated, because FS1 can interfere with the signal interpretation as can the presence of extraneous background iron–sulfur clusters. It has previously been shown, however, that the NarH–FS4 species is by far the predominant species and that only at low potentials does FS1 begin to interfere. ,,, The nearest signal due to FS1 appears at g = 2.013, and the background signal exhibits a peak at 2.02, which overlaps with the dominant signal of FS4 at 2.021.…”

“…However, the two subpopulations collapse into one signal, albeit with a shifted g z , upon inhibitor binding. In the case of HOQNO, PCP, and stigmatellin binding, the g z value for heme b D shifts from 3.35 to 3.50, 3.45, and 3.31, respectively. − Likewise, the NarGHI K86A Q-site variant not only exhibits a homogeneous heme b D EPR signal at ∼3.3 but also exhibits reduced quinol oxidase activity and is unable to bind HOQNO or stabilize a semiquinone radical. ,, Using the inhibitor-bound states also addresses the possibility, and likely inevitability, of interactions of the adjacent heme ( b D ) with the bound quinone species, particularly the stable anionic semiquinone. , For the reasons described above, we focus our analyses herein on the more homogeneous inhibitor-bound states and the NarGHI K86A Q-site variant.…”