A conserved lysine residue controls iron–sulfur cluster redox chemistry in Escherichia coli fumarate reductase

Abstract

The Escherichia coli respiratory complex II paralogs succinate dehydrogenase (SdhCDAB) and fumarate reductase (FrdABCD) catalyze interconversion of succinate and fumarate coupled to quinone reduction or oxidation, respectively. Based on structural comparison of the two enzymes, equivalent residues at the interface between the highly homologous soluble domains and the divergent membrane anchor domains were targeted for study. This included the residue pair SdhB-R205 and FrdB-S203, as well as the conserved SdhB-K230 and FrdB-K228 pair. The close proximity of these residues to the [3Fe-4S] cluster and the quinone binding pocket provided an excellent opportunity to investigate factors controlling the reduction potential of the [3Fe-4S] cluster, the directionality of electron transfer and catalysis, and the architecture and chemistry of the quinone binding sites. Our results indicate that both SdhB-R205 and SdhB-K230 play important roles in fine tuning the reduction potential of both the [3Fe-4S] cluster and the heme. In FrdABCD, mutation of FrdB-S203 did not alter the reduction potential of the [3Fe-4S] cluster, but removal of the basic residue at FrdB-K228 caused a significant downward shift (>100mV) in potential. The latter residue is also indispensable for quinone binding and enzyme activity. The differences observed for the FrdB-K228 and Sdh-K230 variants can be attributed to the different locations of the quinone binding site in the two paralogs. Although this residue is absolutely conserved, they have diverged to achieve different functions in Frd and Sdh.

Redox Behavior of the S-Adenosylmethionine (SAM)-Binding Fe–S Cluster in Methylthiotransferase RimO, toward Understanding Dual SAM Activity

“…Among them, arginine 268 (R268) looked promising for further studies, as it is strictly conserved in all RimO orthologs sequenced to date and interacts directly with several amino acid residues close to the RS cluster (Figure ). Moreover, examples in the literature show that positively charged residues residing near an Fe–S cluster have a strong impact on the cluster redox potential . The Tm -RimO­(R268A) mutant was therefore produced, purified, and reconstituted to give a protein sample with expected analytical properties very similar to those of the WT protein (Table S2 and Figure S1).…”

“…Moreover, examples in the literature show that positively charged residues residing near an Fe−S cluster have a strong impact on the cluster redox potential. 30 The Tm-RimO(R268A) mutant was therefore produced, purified, and reconstituted to give a protein sample with expected analytical properties very similar to those of the WT protein (Table S2 and Figure S1). Interestingly, this mutant was found to be totally inactive in the MTTase reaction.…”

Redox State of Flavin Adenine Dinucleotide Drives Substrate Binding and Product Release in Escherichia coli Succinate Dehydrogenase

“…All recombinant plasmids were verified by DNA sequencing. Variant enzymes, isolated as the major component of the cytoplasmic membrane fraction, were malonate-activated as previously described. , Additionally, the membranes were subsequently pelleted by centrifugation at 100000 g and resuspended in either 100 mM MOPS/5 mM EDTA (pH 7) or 100 mM tricine/5 mM EDTA (pH 8) to remove the malonate.…”

Insights into the catalysis of a lysine-tryptophan bond in bacterial peptides by a SPASM domain radical S-adenosylmethionine (SAM) peptide cyclase

“…These non-cysteine ligands could be used by SPASM domain radical SAM enzymes to finely tune the redox properties of the [4Fe-4S] clusters, as shown for fumarate reductase ( 32 ) or glutaredoxins ( 33 ). However, after AlbA ( 6 ) and now the KW_cyclase, it is likely that a growing number of SPASM domain radical SAM enzymes will exhibit a similar type of [4Fe-4S] cluster coordination.…”