The iron-sulfur clusters in Escherichia coli succinate dehydrogenase direct electron flow.
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.
Cheng
Victor W T
VW
Membrane Protein Research Group, Department of Biochemistry, University of Alberta, 473 Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada.
Ma
Elysia
E
Zhao
Zhongwei
Z
Rothery
Richard A
RA
Weiner
Joel H
JH
D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology*; D005650:Fumarates / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010957:Plasmids / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017382:Reactive Oxygen Species; D013385:Succinate Dehydrogenase / Q000737:chemistry*; D014451:Ubiquinone / Q000737:chemistry
D005650:Fumarates; D007506:Iron-Sulfur Proteins; D017382:Reactive Oxygen Species; D014451:Ubiquinone; C032005:fumaric acid; D013385:Succinate Dehydrogenase