Aspartate 141 is the fourth ligand of the oxygen-sensing [4Fe-4S]2+ cluster of Bacillus subtilis transcriptional regulator Fnr

Abstract

The Bacillus subtilis redox regulator Fnr controls genes of the anaerobic metabolism in response to low oxygen tension. An unusual structure for the oxygen-sensing [4Fe-4S](2+) cluster was detected by a combination of genetic experiments with UV-visible and Mössbauer spectroscopy. Asp-141 was identified as the fourth iron-sulfur cluster ligand besides three Cys residues. Exchange of Asp-141 with Ala abolished functional in vivo complementation of an fnr knock-out strain by the mutagenized fnr gene and in vitro DNA binding of the recombinant regulator FnrD141A. In contrast, substitution of Asp-141 with Cys preserved [4Fe-4S](2+) structure and regulator function.

FIGURE 1.

Functional analyses of B. subtilis Fnr and mutant derivatives for in vivo complementation of a mutant fnr strain and in vitro promoter binding. A, shown are relevant genomic structures of B. subtilis strain HRB3 used for in vivo complementation experiments. The chromosomal fnr copy is inactivated by insertion of a spectinomycin resistance cassette. The Fnr-dependent narG promoter is fused to the reporter gene lacZ, and the tested fnr gene integrated into the amyE locus is expressed under the control of the xylA promoter. B, Fnr function was monitored by expression of an Fnr-dependent narG-lacZ reporter gene fusion under nitrate respiratory growth conditions. Corresponding β-galactosidase activities are given in Miller units. C, a DNase I footprint experiment using a narG promoter fragment representing the coding strand was performed without the addition of protein (lane 1) or with the addition of 1.6 μm FnrCCC (lane 2), FnrD63A (lane 3), and FnrD141A (lane 4). DNA sequencing ladders were included to localize the Fnr-binding sites. The white bar indicates the protected region, and the DNase I-hypersensitive site is marked by an asterisk.

FIGURE 2.

Spectroscopic analysis of B. subtilis Fnr variants. A, UV-visible spectra of anoxically purified B. subtilis wild-type Fnr (solid line), FnrD63A (dotted line), and FnrD141A (dashed line). B, Mössbauer spectra recorded at 77 k of B. subtilis wild-type Fnr (panel I), FnrD141A (panel II), and FnrD141C (panel III) in whole cell extracts of producing E. coli cells. The results of least square fit analyses represented by the solid traces of the subspectra are described in the text, and parameters are given in Table 1.

FIGURE 3.

Proposed location and function of the [4Fe-4S]²⁺ cluster using a structural model of B. subtilis Fnr. A, sequence and structure comparison of B. subtilis Fnr with L. monocytogenes PrfA and E. coli Crp. Identical residues are shaded in gray, and ligands of the [4Fe-4S]²⁺ cluster are indicated in red. The secondary structure elements of PrfA and Crp are schematically shown above and below the alignment: α-helices are represented as lines and β-strands as arrows. B, model of the B. subtilis Fnr monomer based on the structure of the constitutively active PrfA mutant PrfA-G145S (Protein Data Bank code 2BGC) using SWISS-MODEL (ExPASy). The C-terminal amino acid residues unique to Fnr are shown as an extension (black line). This region contains three cysteine residues (Cys-227, Cys-230, and Cys-235) coordinating the [4Fe-4S]²⁺ cluster together with Asp-141. C, model of [4Fe-4S]²⁺ cluster-dependent activation of Fnr. Under aerobic conditions (+ O₂), dimeric Fnr does not carry an intact [4Fe-4S]²⁺ cluster. Anoxic conditions allow FeS cluster formation with subsequent movement of helix D. This novel conformation is the prerequisite for promoter binding. (The N-terminal part of Fnr is not represented in the model.)