Superoxide-mediated amplification of the oxygen-induced switch from [4Fe-4S] to [2Fe-2S] clusters in the transcriptional regulator FNR

Abstract

In Escherichia coli, the switch between aerobic and anaerobic metabolism is controlled primarily by FNR (regulator of fumarate and nitrate reduction), the protein that regulates the transcription of >100 genes in response to oxygen. DNA regulation ͉ iron-sulfur I n Escherichia coli, the switch between aerobic and anaerobic respiration is primarily controlled by the transcriptional regulator of fumarate and nitrate reduction (FNR) (1-3). The protein belongs to a large family of regulators that modulate physiological changes in response to various environmental and metabolic challenges (4-6). Together with the E. coli cAMP receptor protein (CRP), FNR is a pivotal member of an expanding superfamily of structurally related transcriptional factors (5). The archetypal CRP structural fold provides a versatile system for transducing either environmental or metabolic signals into a physiological response (5-7). Based on sequence homology, FNR, like CRP, consists of two distinct domains that provide DNA-binding and sensory functions (see Fig. 1) (7-9). The C-terminal DNA-binding domain recognizes specific FNRbinding sequences within FNR-controlled promoters (10). The N-terminal sensory domain contains five cysteine residues, four of which (12,(14)(15)(16)(18)(19)(20).The mechanism of the oxygen-mediated cluster conversion is of considerable current interest. Various mechanisms have been proposed for this process, including oxygen reduction to hydrogen peroxide through metal-centered oxidation (15) and oxygen reduction to water through sulfide-based oxidation (21). Recently, we reported the detection of approximately two sulfide ions released per FNR monomer during cluster conversion (22), demonstrating that cluster oxidation is metal based.Here, we demonstrate that the reaction of oxygen with [4Fe-4S] FNR (either native or reconstituted) in vitro occurs in two steps. The first is a second-order, one-electron oxidation of the [4Fe-4S] 2ϩ cluster, leading to the generation of superoxide ion and a [3Fe-4S] 1ϩ cluster intermediate, with the ejection of one Fe 2ϩ . The second step is the spontaneous (first-order) conversion of the [3Fe-4S] 1ϩ cluster to the [2Fe-2S] 2ϩ form, with the release of two sulfides and a Fe 3ϩ ion. Superoxide generated during the first step undergoes, at least in part, dismutation to hydrogen peroxide and oxygen. We propose that catalytic recycling of superoxide/hydrogen peroxide back to oxygen provides a means to amplify the sensitivity of [4Fe-4S] FNR to its signal. Results Characterization of Intermediates During the Oxidation of FNR.We previously reported the detection of an EPR-active species, withAuthor contributions: J.C.C., J.G., M.R.C., N.E.L.B., and A.J.T. designed research; J.C.C. performed research; J.C.C. and N.E.L.B. analyzed data; and J.C.C., J.G., N.E.L.B., and A.J.T. wrote the paper.The authors declare no conflict of interest.This article is a PNAS direct submission.Abbreviations: FNR, regulator of fumarate and nitrate reduction; Fd, ferredoxin; Ferene, 5,5Ј(3-(2-pyridyl)-1,2,4-tri...

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