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 (bD and bP) 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 bp and the [3Fe-4S] cluster exhibited two apparent components; (ii) heme bD 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 bD. 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 Q-site in the vicinity of heme bP. In a re-examination of the redox behavior of hemes bD and bP 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.