1p2h
H61M mutant of flavocytochrome c3
Structural highlights
FunctionFCCA_SHEFR Flavocytochrome that catalyzes the reduction of fumarate to succinate (PubMed:10978153). Is essential for fumarate respiration during anaerobic growth, acting as the terminal reductase (By similarity). Receives electrons from the membrane-bound tetraheme c-type cytochrome CymA (By similarity). In vitro, can use the artificial electron donor methyl viologen (PubMed:10978153).[UniProtKB:P83223][UniProtKB:Q07WU7][1] Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedAn examination of the X-ray structure of the soluble fumarate reductase from Shewanella frigidimarina [Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112] shows the presence of four, bis-His-ligated, c-type hemes and one flavin adenine dinucleotide, FAD. The heme groups provide a "molecular wire" for the delivery of electrons to the FAD. Heme IV is closest to the FAD (7.4 A from heme methyl to FAD C7), and His61, a ligand to heme IV, is also close (8.4 A to FAD C7). Electron delivery to the FAD from the heme groups must proceed via heme IV, as hemes I-III are too far from the FAD for feasible electron transfer. To examine the importance of heme IV and its ligation for enzyme function, we have substituted His61 with both methionine and alanine. Here we describe the crystallographic, kinetic, and electrochemical characterization of the H61M and H61A mutant forms of the Shewanella fumarate reductase. The crystal structures of these mutant forms of the enzyme have been determined to 2.1 and 2.2 A resolution, respectively. Substitution of His61 with alanine results in heme IV having only one protein ligand (His86), the sixth coordination position being occupied by an acetate ion derived from the crystal cryoprotectant solution. In the structure of the H61M enzyme, Met61 is found not to ligate the heme iron, a role that is taken by a water molecule. Apart from these features, there are no significant structural alterations as a result of either substitution. Both the H61M-Fcc(3) and H61A-Fcc(3) mutant enzymes are catalytically active but exhibit marked decreases in the value of k(cat) for fumarate reduction with respect to that of the wild type (5- and 10-fold lower, respectively). There is also a significant shift in the pK(a) values for the mutant enzymes, from 7.5 for the wild type to 8.26 for H61M and 9.29 for H61A. The fumarate reductase activity of both mutant enzymes can be recovered to approximately 80% of that seen for the wild type by the addition of exogenous imidazole. In the case of H61A, recovery of activity is also accompanied by a shift of the pK(a) from 9.29 to 7.46 (close, and within experimental error, to that for the wild type). Pre-steady-state kinetic measurements show clearly that rate constants for the fumarate dependent reoxidation of the heme groups are adversely affected by the mutations. The solvent isotope effect for fumarate reduction in the wild-type enzyme has a value of 8.0, indicating that proton delivery is substantially rate limiting. This value falls to 5.6 and 2.2 for the H61M and H61A mutants, respectively, indicating that electron transfer, rather than proton transfer, is becoming more rate-limiting in the mutant enzymes. Histidine 61: an important heme ligand in the soluble fumarate reductase from Shewanella frigidimarina.,Rothery EL, Mowat CG, Miles CS, Walkinshaw MD, Reid GA, Chapman SK Biochemistry. 2003 Nov 18;42(45):13160-9. PMID:14609326[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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