SummaryThe oxygen regulator Fnr is part of the regulatory cascade in Bacillus subtilis for the adaptation to anaerobic growth conditions. In vivo complementation experiments revealed the essential role of only three cysteine residues (C227, C230, C235) at the Cterminus of B. subtilis Fnr for the transcriptional activation of the nitrate reductase operon ( narGHJI ) and nitrite extrusion protein gene ( narK ) promoters. UV/ VIS, electron paramagnetic spin resonance (EPR) and Mössbauer spectroscopy experiments in combination with iron and sulphide content determinations using anaerobically purified recombinant B. subtilis Fnr identified the role of these three cysteine residues in the formation of one
Eukaryotic life is adapted to an oxygen-dependent energy generation. In contrast, many prokaryotes populate their ecological niches by the ability to survive and multiply under conditions of low oxygen tension. Anaerobic growth of these organisms is mediated by a broad spectrum of proton gradient-generating alternative respiration strategies. In addition, substrate level phosphorylation employing highly diverse fermentation processes allows for ATP formation and growth under anaerobic conditions. Moreover, this anaerobic energy metabolism was found to be essential for many pathogenic microorganisms during infection and biofilm formation. Because of the better energy yield, organisms prefer oxygen-dependent growth over anaerobic energy generation strategies. Additionally, several components of the anaerobic respiratory machineries were found to be oxygen-sensitive. Consequently, a strict regulation of the aerobic-anaerobic transition is advised. For oxygen sensing, many different types of sensors have been identified in bacteria and archaea, most of which react directly with oxygen by oxygen-reactive groups, like heme, FeS clusters, cysteine pairs, and FAD (1).A key regulatory protein in bacteria is Fnr, named after the fumarate and nitrate reduction-negative phenotype of an fnr gene-defective Escherichia coli strain (2). The N-terminal part of the E. coli Fnr protein contains four cysteine residues, Cys-20, Cys-23, Cys-29, and Cys-122, which are essential for Fnr function by coordinating a [4Fe-4S] 2ϩ cluster responsible for oxygen sensing. The C-terminal DNA-binding domain recognizes specific binding sites located in Fnr-controlled promoters (2).In contrast to E. coli Fnr, the [4Fe-4S] 2ϩ cluster of the redox regulator from the Gram-positive model bacterium Bacillus subtilis possesses only three cysteine ligands localized at the C terminus of the protein and a fourth unknown non-cysteinyl ligand (3). In most cases, the iron atoms of 2ϩ clusters are coordinated by four cysteine residues as found for E. coli Fnr. However, there are reported precedents of iron-sulfur clusters with only three cysteine ligands. Histidine residues are known to be involved in iron-sulfur cluster coordination in so-called "Rieske clusters" that are usually part of membrane-localized electron-transferring proteins (4). However, alanine substitutions of all conserved histidine residues of B. subtilis Fnr failed to abolish Fnr function and excluded histidines as the fourth ligand (3).
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