Abstract
Succinate dehydrogenase is an indispensable enzyme involved in the Krebs cycle as well as energy coupling in the mitochondria and certain prokaryotes. During catalysis, succinate oxidation is coupled to ubiquinone reduction by an electron transfer relay comprising a flavin adenine dinucleotide cofactor, three iron-sulfur clusters, and possibly a heme b556. At the heart of the electron transport chain is a [4Fe-4S] cluster with a low midpoint potential that acts as an energy barrier against electron transfer. Hydrophobic residues around the [4Fe-4S] cluster were mutated to determine their effects on the midpoint potential of the cluster as well as electron transfer rates. SdhB-I150E and SdhB-I150H mutants lowered the midpoint potential of this cluster; surprisingly, the His variant had a lower midpoint potential than the Glu mutant. Mutation of SdhB-Leu-220 to Ser did not alter the redox behavior of the cluster but instead lowered the midpoint potential of the [3Fe-4S] cluster. To correlate the midpoint potential changes in these mutants to enzyme function, we monitored aerobic growth in succinate minimal medium, anaerobic growth in glycerol-fumarate minimal medium, non-physiological and physiological enzyme activities, and heme reduction. It was discovered that a decrease in midpoint potential of either the [4Fe-4S] cluster or the [3Fe-4S] cluster is accompanied by a decrease in the rate of enzyme turnover. We hypothesize that this occurs because the midpoint potentials of the [Fe-S] clusters in the native enzyme are poised such that direction of electron transfer from succinate to ubiquinone is favored.
Highlights
Succinate dehydrogenase (Sdh, Complex II in eukaryotes) is an indispensable enzyme involved in the Krebs cycle and aerobic respiration
Based on results obtained in this study, we propose a novel role for [Fe-S] clusters in ET relays whereby their midpoint potentials dictate the direction of electron transfer
We first examined whether mutation of a hydrophobic residue to a charged or polar residue in the middle of the electron transfer subunit had an impact on the proper assembly and targeting of Sdh
Summary
Bacterial Strains and Plasmids—E. coli strain DW35 (⌬frdABCD, sdhC::kan) [28] was used for all enzyme expression and growth studies. Malonate was added to a final concentration of 1 mM, and membranes were incubated at 30 °C for 15 min to activate the Sdh enzyme. Redox Titration and EPR Spectroscopy—Redox titrations were carried out anaerobically under argon at 25 °C on Sdhenriched membranes at a total protein concentration of ϳ30 mg mlϪ1 in 50 mM MOPS/5 mM EDTA, pH 7.0. Anaerobic succinate-dependent reduction of Q0 (⑀ ϭ 0.73 mMϪ1 cmϪ1) was monitored at 410 nm; anaerobicity was achieved by saturating the assay buffer with N2 and the addition of glucose (20 mM) and glucose oxidase (8.8 units mlϪ1) to the reaction cuvette. Sdh-enriched membranes at a total concentration of 1 mg mlϪ1 in N2-saturated 100 mM MOPS/5 mM EDTA, pH 7.0, buffer were used. Sdh enzyme, cytochrome c, Q0, KCN, superoxide dismutase, and succinate were added in the order listed to final concentrations of 1 g mlϪ1, 10 M, 50 M, 10 mM, 30 units mlϪ1, and 10 mM, respectively
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