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J. Biol. Chem., Vol. 276, Issue 4, 2432-2439, January 26, 2001

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Characterization of the Intramolecular Electron Transfer Pathway from 2-Hydroxyphenazine to the Heterodisulfide Reductase from Methanosarcina thermophila^*

Eisuke Murakami, Uwe Deppenmeier^§, and Stephen W. Ragsdale^¶

From the  Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln, Nebraska 68588-0664 and ^§ Institut Für Mikrobiologie, Der Georg-August-Universität Göttingen, Grisebachstrae 8, Göttingen D-37077, Germany

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 [Fe₄S₄] 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 (HPhenH₂) to CoB-S-S-CoM. Steady-state kinetic studies indicate a ping-pong mechanism for heterodisulfide reduction by HPhenH₂ with the following values: k_cat = 74 s¹ at 25 °C, K_m (HPhenH₂) = 92 µM, K_m (CoB-S-S-CoM) = 144 µM. 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 HPhenH₂. Fe-S cluster disruption by mersalyl acid inhibits heme reduction by HPhenH₂, suggesting that a 4Fe cluster is the initial electron acceptor from HPhenH₂. We propose the following electron transfer pathway: HPhenH₂ to the high potential 4Fe cluster, to the low potential heme, and finally, to CoB-S-S-CoM.

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