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

We have investigated the role of redox cooperativity in defining the functional relationship among the three membrane-associated prosthetic groups of Escherichia coli nitrate reductase A: the two hemes (b(D) and b(p)) of the membrane anchor subunit (NarI) and the [3Fe-4S] cluster (FS4) of the electron-transfer subunit (NarH). Previously published analyses of potentiometric titrations have exhibited the following anomalous behaviors: (i) fits of titration data for heme b(p) and the [3Fe-4S] cluster exhibited two apparent components; (ii) heme b(D) titrated with an apparent electron stoichiometry (n) of <1.0; and (iii) the binding of quinol oxidation inhibitors shifted the reduction potentials of both hemes despite there being only a single quinol oxidation site (Q-site) in close juxtaposition with heme b(D). Furthermore, both hemes appeared to be affected despite the absence of major structural shifts upon inhibitor binding, as judged by X-ray crystallography, or evidence of a second Qsite in the vicinity of heme b(p). In a re-examination of the redox behavior of hemes b(D) and b(p) and FS4, we have developed a cooperative redox model of cofactor interaction. We show that anticooperative interactions provide an explanation for the anomalous behavior. We propose that the role of such anticooperative redox behavior in vivo is to facilitate transmembrane electron transfer across an energy-conserving membrane against an electrochemical potential.