title: Characterization of the intramolecular electron transfer pathway from 2-hydroxyphenazine to the heterodisulfide reductase from Methanosarcina thermophila.
abstract: Heterodisulfide reductase (HDR) is a component of the energy-conserving electron transfer system in methanogens. HDR catalyzes the two-electron reduction of coenzyme B-S-S-coenzyme M (CoB-S-S-CoM), the heterodisulfide product of the methyl-CoM reductase reaction, to free thiols, HS-CoB and HS-CoM. HDR from Methanosarcina thermophila contains two b-hemes and two [Fe(4)S(4)] clusters. The physiological electron donor for HDR appears to be methanophenazine (MPhen), a membrane-bound cofactor, which can be replaced by a water-soluble analog, 2-hydroxyphenazine (HPhen). This report describes the electron transfer pathway from reduced HPhen (HPhenH(2)) to CoB-S-S-CoM. Steady-state kinetic studies indicate a ping-pong mechanism for heterodisulfide reduction by HPhenH(2) with the following values: k(cat) = 74 s(-1) at 25 degrees C, K(m) (HPhenH(2)) = 92 microm, K(m) (CoB-S-S-CoM) = 144 microm. Rapid freeze-quench EPR and stopped-flow kinetic studies and inhibition experiments using CO and diphenylene iodonium indicate that only the low spin heme and the high potential FeS cluster are involved in CoB-S-S-CoM reduction by HPhenH(2). Fe-S cluster disruption by mersalyl acid inhibits heme reduction by HPhenH(2), suggesting that a 4Fe cluster is the initial electron acceptor from HPhenH(2). We propose the following electron transfer pathway: HPhenH(2) to the high potential 4Fe cluster, to the low potential heme, and finally, to CoB-S-S-CoM.
authors.affiliation: Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln 68588-0664, USA.
authors.forename: E
authors.initials: E
authors.lastname: Murakami
authors.forename: U
authors.initials: U
authors.lastname: Deppenmeier
authors.forename: S W
authors.initials: SW
authors.lastname: Ragsdale
mesh_terms: D002248:Carbon Monoxide / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D017022:Flow Injection Analysis; D007506:Iron-Sulfur Proteins / Q000187:drug effects / Q000378:metabolism*; D008634:Mersalyl / Q000494:pharmacology; D015080:Mesna / Q000378:metabolism; D017020:Methanosarcina / Q000201:enzymology*; D009861:Onium Compounds / Q000494:pharmacology; D010088:Oxidoreductases / Q000187:drug effects / Q000378:metabolism*; D010619:Phenazines / Q000378:metabolism*; D010769:Phosphothreonine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D007506:Iron-Sulfur Proteins; D009861:Onium Compounds; D010619:Phenazines; C049349:7-mercaptoheptanoylthreonine phosphate; D010769:Phosphothreonine; C088013:2-hydroxyphenazine; D008634:Mersalyl; C007517:diphenyleneiodonium; D002248:Carbon Monoxide; D010088:Oxidoreductases; C087171:heterodisulfide reductase; D015080:Mesna