Characterization of the Intramolecular Electron Transfer Pathway from 2-Hydroxyphenazine to the Heterodisulfide Reductase fromMethanosarcina thermophila

Eisuke Murakami,Uwe Deppenmeier,Stephen W Ragsdale

doi:10.1074/jbc.m004809200

Eisuke Murakami, Uwe Deppenmeier + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m004809200

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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.

Highlights

Heterodisulfide reductase (HDR)1 plays important roles in methane production by methanogenic archaea
Which of the metal centers in HDR is the initial electron acceptor from reduced HPhen (HPhenH2)? The midpoint potentials of some of the metal centers are outside the range of the HPhen/HPhenH2 and CoB-S-S-CoM/CoB-SH, CoM-SH couples; are all of the metal clusters involved in the electron transfer reaction? What is the intramolecular electron transfer pathway? Based on our results, we propose that the physiological electron transfer pathway from methanophenazine to the heterodisulfide is: MPhenH2 3 [Fe4S4]high 3 hemelow 3 CoB-S-S-CoM
Steady State—Steady-state kinetic experiments were performed at 55 °C at varying concentrations of HPhenH2 and CoB-S-S-CoM to determine the overall mechanism of the HDR reaction

Summary

EXPERIMENTAL PROCEDURES

Materials—HPhen was synthesized as described [11]. Other chemicals were purchased from Sigma Chemical Co. HDR was purified as described previously [6], except for enzyme concentration steps. Enzyme Assays—HDR activity was measured by monitoring the oxidation of reduced methyl viologen at 604 nm (⑀ ϭ 13.9 mMϪ1 cmϪ1) and 55 °C [6, 17, 18]. One unit of HDR activity corresponds to 1 ␮mol of CoB-S-S-CoM reduced per minute. A solution containing HPhen (final concentration of 200 ␮M) in Buffer A (50 mM Tris, pH 7.6, and 10% glycerol) was reduced by bubbling with 100% hydrogen gas for 20 min, adding partially purified hydrogenase from M. thermophila, and incubating at 55 °C for 2 h. The data were fit to eq 3 for a ping-pong reaction, where v0 is initial velocity and A and B are HPhenH2 and CoB-S-S-CoM, respectively, as follows.

Electron Transfer in Heterodisulfide Reductase

RESULTS

Ox Red

DISCUSSION

Why would HDR retain an unnecessary high potential heme