The metal‐free hydrogenase from methanogenic archaea: evidence for a bound cofactor

Buurman, Gerrit; Shima, Seigo; Thauer, Rudolf K.

doi:10.1016/s0014-5793(00)02225-0

Cited by 92 publications

(84 citation statements)

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“…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.…”

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confidence: 83%

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

Korbas

Vogt

Meyer‐Klaucke

et al. 2006

Journal of Biological Chemistry

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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 Cys 176 , 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 H 2 interaction.Hydrogenases are enzymes that catalyze the reversible oxidation of molecular hydrogen (1). Their structure and catalytic mechanism are of considerable applied interest as models for the development of efficient catalysts for hydrogen-fueled processes. Despite intensive efforts, however, the understanding of how hydrogenases react with H 2 is only in its infancy.Two of the three known types of hydrogenases are ironsulfur proteins that contain, besides one or several iron-sulfur clusters, a dinuclear metal center, either [NiFe] or [FeFe], which was shown to be the site of H 2 reaction. Both dinuclear hydrogenases catalyze the reversible formation of 2e Ϫ and 2Hϩ from H 2 , the electrons being transferred one by one to electron acceptors via the iron-sulfur clusters. One iron ion in both dinuclear centers is coordinated by sulfur, CO, and cyanide ligands, as supported by crystal structures of the [NiFe]-hydrogenase (2, 3) and of the [FeFe]-hydrogenase (4 -7). During the catalytic cycle, the iron ion in the [NiFe] center remains in a low spin ferrous state and is not redoxactive (8, 9). In the case of the [FeFe]-hydrogenases, the oxidation states of the dinuclear iron center are still under discussion; however, the involvement of a low spin Fe(II) has been predicted based on the spectroscopic and DFT studies (9 -11). Despite similarities in structures and properties, [FeFe]-and [NiFe]-hydrogenases are phylogenetically not related.[FeFe]-hydrogenases are found in bacteria and eucarya, whereas [NiFe]-hydrogenases are found in bacteria and archaea. For recent reviews on these two types of hydrogenases, see Frey (9), Zhou et al.

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“…The apoprotein is inactive. 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). Similarly to the [NiFe]-hydrogenases, the iron ion in Hmd is not redox-active.…”

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confidence: 99%

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

Korbas

Vogt

Meyer‐Klaucke

et al. 2006

Journal of Biological Chemistry

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136

162

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“…In this reaction, a hydride is transferred from H 2 into the pro-R position of the methylene carbon of methylene-H 4 MPT [3,6] (Chart 1). Although it was previously thought to be metalfree [4], Hmd is now known to harbor a mononuclear iron containing cofactor of unknown structure that is essential for activity [7,8].…”

Section: Introductionmentioning

confidence: 99%

Characterization of the Fe Site in Iron−Sulfur Cluster-Free Hydrogenase (Hmd) and of a Model Compound via Nuclear Resonance Vibrational Spectroscopy (NRVS)

et al. 2008

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We have used 57 Fe nuclear resonance vibrational spectroscopy (NRVS) to study the iron site in the iron-sulfur-cluster-free hydrogenase Hmd from the methanogenic archaeon Methanothermobacter marburgensis. The spectra have been interpreted by comparison with a cis-(CO) 2 -ligated Fe model compound, Fe(S 2 C 2 H 4 )(CO) 2 (PMe 3 ) 2 , as well as by normal mode simulations of plausible active site structures. For this model complex, normal mode analyses both from an optimized Urey-Bradley force field and from complementary density functional theory (DFT) calculations produced consistent results.Previous IR spectroscopic studies found strong CO stretching modes at 1944 and 2011 cm −1 , interpreted as evidence for cis-Fe(CO) 2 ligation. The NRVS data provide further insight into the dynamics of the Fe site, revealing Fe-CO stretch and Fe-CO bend modes at 494, 562, 590, and 648 cm −1 , consistent with the proposed cis-Fe(CO) 2 ligation. The NRVS also reveals a band assigned to Fe-S stretching motion at ~311 cm −1 , and another reproducible feature at ~380 cm −1 . The 57 Fe partial vibrational densities of states (PVDOS) for Hmd can be reasonably well simulated by a normal mode analysis based on a Urey-Bradley force field for a 5-coordinate cis-(CO) 2 -ligated Fe site with additional cysteine, water, and pyridone cofactor ligands. A final interpretation of the Hmd NRVS data, including DFT analysis, awaits a 3-dimensional structure for the active site.

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“…It does not mediate the reduction of dyes such as methylviologen or methylene blue nor the exchange of protons with hydrogen isotopes (unless the substrate methenyl-H 4 MPT + is present) (Schwörer et al, 1993). It was shown recently to bind a cofactor, which can reconstitute active Hmd from the apoenzyme (Buurman et al, 2000). The enzyme and the cofactor are sensitive to UV-A (320-400 nm) or blue (400-500 nm) light.…”

Section: The Iron-sulfur Cluster Free-hydrogenases Hmdmentioning

confidence: 99%

Molecular Biology of Microbial Hydrogenases

2004

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The metal‐free hydrogenase from methanogenic archaea: evidence for a bound cofactor

Cited by 92 publications

References 22 publications

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

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

Characterization of the Fe Site in Iron−Sulfur Cluster-Free Hydrogenase (Hmd) and of a Model Compound via Nuclear Resonance Vibrational Spectroscopy (NRVS)

Molecular Biology of Microbial Hydrogenases

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