Characterization of the intramolecular electron transfer pathway from 2-hydroxyphenazine to the heterodisulfide reductase from Methanosarcina thermophilo

Heterodisulfide reductase (HDR) is a component of the energy-conserving ele ctron transfer system in methanogens, HDR catalyzes the two-electron reduct ion 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, H DR from Methanosarcina thermophila contains two b-hemes and two [Fe4S4] clu sters. The physiological electron donor for HDR appears to be methanophenaz ine (MPhen), a membrane-bound cofactor, which can be replaced by a water-so luble analog, 2-hydroxyphenazine (HPhen), This report describes the electro n transfer pathway from reduced HPhen (HPhenH(2)) to CoB-S-S-CoM. Steady-st ate kinetic studies indicate a ping-pong mechanism for heterodisulfide redu ction by HPhenH(2) with the following values: k(cat) = 74 s(-1) at 25 degre esC, K-m (HPhenH(2)) = 92 muM, K-m (CoB-S-S-CoM) = 144 muM Rapid freeze-que nch EPR and stopped-flow kinetic studies and inhibition experiments using C O and diphenylene iodonium indicate that only the low spin heme and the hig h 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 HP henH(2). We propose the following electron transfer pathway: HPhenH(2) to t he high potential 4Fe cluster, to the low potential heme, and finally, to C oB-S-S-CoM.