Modulation of Fe‐S cluster midpoint potential and electron transfer rates in Escherichia coli succinate dehydrogenase

Abstract

Succinate Dehydrogenase (Sdh, Complex II) is an indispensable enzyme involved in the Krebs cycle as well as energy coupling in the mitochondria and certain prokaryotes. During enzyme turnover, succinate is oxidized to fumarate in the catalytic subunit (SdhA) and donates two electrons to a flavin adenine dinucleotide cofactor. The electrons are then shuttled singly through a series of iron-sulfur (Fe-S) clusters in SdhB until they reach the membrane anchor domain (SdhCD), where they reduce ubiquinone to ubiquinol. At the heart of the electron transport chain is a [4Fe-4S] cluster (FS2) with a low midpoint potential (Eo) that acts as an energy barrier against electron transfer. Hydrophobic residues around FS2 were mutated to determine their effects on Fe-S cluster electrochemistry as well as electron transfer rates. SdhB-I150E and SdhB-I150H mutants lowered the Eo of FS2; surprisingly, the His variant had a lower Eo than the Glu mutant. Mutation of SdhB-L220 to His had no effect, but mutation to Asp lowered the Eo of FS2. More interestingly, converting SdhB-L220 to Ser did not alter the electrochemistry of FS2 but instead lowered the Eo of the [3Fe-4S] cluster. To corroborate the Eo changes in these mutants to enzyme function, numerous assays were performed. These included aerobic growth in succinate minimal media, anaerobic growth in glycerol-fumarate minimal media, non-physiological succinate:PMS-MTT enzyme assay, physiological succinate:Q0 enzyme assay, and heme reduction assay. In general, it was discovered that a decrease in Eo of either the [4Fe-4S] cluster or the [3Fe-4S] cluster is accompanied by a decrease in the rate of enzyme turnover. This research is funded by the AHFMR and CIHR.