The Iron-Sulfur Cluster-free Hydrogenase (Hmd) Is a Metalloenzyme with a Novel Iron Binding Motif

Malgorzata Korbas,Sonja Vogt,Wolfram Meyer-Klaucke,Eckhard Bill,Erica J Lyon,Rudolf K Thauer,Seigo Shima

doi:10.1074/jbc.m605306200

Abstract

The iron-sulfur cluster-free hydrogenase (Hmd) from methanogenic archaea harbors an iron-containing cofactor of yet unknown structure. X-ray absorption spectroscopy of the active, as isolated enzyme from Methanothermobacter marburgensis (mHmd) and of the active, reconstituted enzyme from Methanocaldococcus jannaschii (jHmd) revealed the presence of mononuclear iron with two CO, one sulfur and one or two N/O in coordination distance. In jHmd, the single sulfur ligand is most probably provided by Cys176, as deduced from a comparison of the activity and of the x-ray absorption and Mössbauer spectra of the enzyme mutated in any of the three conserved cysteines. In the isolated Hmd cofactor, two CO, one sulfur, and two nitrogen/oxygen atoms coordinate the iron, the sulfur ligand being most probably provided by mercaptoethanol, which is absolutely required for the extraction of the iron-containing cofactor from the holoenzyme and for the stabilization of the extracted cofactor. In active mHmd holoenzyme, the number of iron ligands increased by one when one of the Hmd inhibitors (CO or KCN) were present, indicating that in active Hmd, the iron contains an open coordination site, which is proposed to be the site of H2 interaction.

Highlights

Hydrogenases are enzymes that catalyze the reversible oxidation of molecular hydrogen [1]
The x-ray absorption spectroscopy study revealed the composition of the iron site in active H4MPT dehydrogenase (Hmd) (Table 1)
The novel iron binding motif of mHmd is constituted by two CO molecules: one cysteic sulfur atom and one oxygen/nitrogen atom originating from the organic skeleton of the Hmd cofactor

Summary

The abbreviations used are

H4MPT,tetrahydromethanopterin; XAS, x-ray absorption spectroscopy; mHmd, M. marburgensis Hmd; jHmd, M. jannaschii Hmd; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MOPS, 4-morpholinepropanesulfonic acid; XANES, X-ray absorption near edge structure; EXAFS, extended x-ray absorption fine structure; FT, Fourier transform; Mes, 4-morpholineethanesulfonic acid. The active enzyme can be reconstituted by the addition of the extracted cofactor to heterologously produced Hmd apoprotein [22], whose crystal structure has recently been determined [23]. Heat inactivation of the iron-containing Hmd cofactor was found to be slowed down considerably in the presence of mercaptoethanol both under oxic and anoxic conditions [22]. These results indicate that in the cofactor at least one of the ligands to iron might be a sulfur atom and that the cofactor might be bound to the protein via Fe–S–Cys bonds. MHmd and jHmd show 65% sequence identity

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION