Kinetics of Membrane‐Bound Nitrate Reductase A from Escherichia Coli with Analogues of Physiological Electron Donors

Abstract

We have compared the steady-state kinetics of wild-type nitrate reductase A and two mutant forms with altered β subunits. To mimic conditions in vivo as closely as possible, we used analogues of the physiological quinols as electron donors and membranes with overexpressed nitrate reductase A in preference to a purified Aβγ complex. With the wild-type enzyme both menadiol and duroquinol supply their electrons for the reduction of nitrate at rates that depend on the square of the quinol concentration, menadiol having the higher catalytic constant. The results as a whole are consistent with a substitutedenzyme mechanism for the reduction of nitrate by the quinols.Kinetic experiments suggest that duroquinol and menadiol deliver their electrons at different sites on nitrate reductase, with cross-inhibition. Menadiol inhibits the duroquinol reaction strongly, suggesting that menaquinol may be the preferred substrate in vivo. To examine whether electron transfer from menadiol and duroquinol for nitrate reduction requires the presence of all of the Fe-S centres, we have studied the steady-state kinetics of mutants with β subunits that lack an Fe-S centre. The loss of the highest-potential Fe-S centre results in an enzyme without menadiol activity, but retaining duroquinol activity; the kinetic parameters are within a factor of two of those of the wild-type enzyme, indicating that this centre is not required for the duroquinol activity. The loss of a low-potential Fe-S centre affects the activity with both quinols: the enzyme is still active but the catalytic constants for both quinols are decreased by about 75%, indicating that this centre is important but not essential for the activity.The existence of a specific site of reaction on nitrate reductase for each quinol, together with the differences in the effects on the two quinols produced by the loss of the Fe-S centre of ϩ80 mV, suggests that the pathways for transfer of electrons from duroquinol and menadiol are not identical.Keywords : nitrate reductase; quinol ; menadiol; duroquinol.A respiratory system constituted by formate dehydrogenase et al., 1989). The A and β subunits are located on the cytoplasmic side of the membrane and form an Aβ complex that binds to the and nitrate reductase is induced in Escherichia coli under anaerobic conditions in the presence of nitrate (Pichinoty, 1969; Ruiz-membrane by interacting with the γ subunit, a very hydrophobic protein embedded in the membrane (Sodergren et al., 1988). Herrera and DeMoss, 1969). It allows the flow of electrons to nitrate from the formate that is produced when pyruvate is conEach subunit carries a different set of redox centres (Blasco verted to acetyl-CoA by pyruvate-formate lyase. The electron et al., 1989 ; Guigliarelli et al., 1992). The 139-kDa A subunit transfer is strongly dependent on the presence of a quinone, and contains the active site for the reduction of nitrate to nitrite and either ubiquinone (a benzoquinone) or menaquinone (a naph-carries a Mo cofactor (molybdopterin guanine dinucleotide)...

Interaction of 2-n-Heptyl-4-Hydroxyquinoline-N-Oxide with Dimethyl Sulfoxide Reductase of Escherichia coli

“…Mutation of His-65 to Arg in DmsC blocks quinol activity and eliminates HOQNO binding. Our finding of only one exchangeable binding site, by direct measurement, differs from studies with fumarate reductase (6 -8) and nitrate reductase (24), where two quinol binding sites have been proposed by indirect methods.…”

Q-Site Occupancy Defines Heme Heterogeneity in Escherichia coli Nitrate Reductase A (NarGHI)

“…44 Because NarGHI preferentially uses MQ, which binds to it with a higher affinity over ubiquinone, and better stabilizes menasemiquinone over ubisemiquinone, one therefore expects the component corresponding to the occupied Q-site conformation to correlate most closely with the presence of MQ. 14,34,45 In the case where MQ is absent ( Figure 2B), we observe the most heterogeneity and greatest contribution from the g = 3.18 component. When both MQ and UQ are present, the heterogeneity is reduced, and the g = 3.34 component becomes more prominent.…”

Cardiolipin-based respiratory complex activation in bacteria

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