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

J Biol Chem. 2001 Jan 26;276(4):2432-9. doi: 10.1074/jbc.M004809200. Epub 2000 Oct 16.


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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Carbon Monoxide / pharmacology
  • Electron Spin Resonance Spectroscopy
  • Electron Transport
  • Flow Injection Analysis
  • Iron-Sulfur Proteins / drug effects
  • Iron-Sulfur Proteins / metabolism*
  • Mersalyl / pharmacology
  • Mesna / metabolism
  • Methanosarcina / enzymology*
  • Onium Compounds / pharmacology
  • Oxidoreductases / drug effects
  • Oxidoreductases / metabolism*
  • Phenazines / metabolism*
  • Phosphothreonine / analogs & derivatives*
  • Phosphothreonine / metabolism


  • Iron-Sulfur Proteins
  • Onium Compounds
  • Phenazines
  • 7-mercaptoheptanoylthreonine phosphate
  • Phosphothreonine
  • 2-hydroxyphenazine
  • Mersalyl
  • diphenyleneiodonium
  • Carbon Monoxide
  • Oxidoreductases
  • heterodisulfide reductase
  • Mesna