Metallocenter assembly of the hydrogenase enzymes
Introduction
Metalloproteins contain either a single metal ion or more intricate centers that can include polynuclear clusters, modified amino acids and exogenous ligands. Inorganic prosthetic groups require an extra degree of cellular control because of the inherent toxicity of the metal ions as well as the complexity of the metal clusters on the protein scaffold. Nature meets these challenges with helper proteins dedicated to handling specific metal ions and the assembly of the metalloenzyme centers [1, 2]. These proteins guarantee delivery of the correct metal even under conditions of low intracellular availability, minimize any potential harmful effects of the inorganic and organic pieces, catalyze the synthesis of any components not readily available, and ensure complete assembly and localized placement of the clusters in the enzyme proteins.
The hydrogenase enzymes, which catalyze the reversible oxidation of hydrogen gas, enable an organism to use molecular hydrogen as a source of energy or to use protons as a sink for excess reducing equivalents [3]. These enzymes are divided into three phylogenetically distinct classes that differ in metal content: [NiFe], [FeFe] and ‘iron–sulfur cluster-free’ [3, 4, 5, 6•]. The latter enzymes, which used to be called ’metal-free’, are now known to contain a mononuclear iron center that has not yet been completely defined ([6•] and references therein), and nothing is yet known about the biosynthesis of this site. By contrast, significant progress has been made in understanding the assembly of the [NiFe] hydrogenase active site, and the accessory proteins for the [FeFe] active site production were recently identified and characterized. This information about the activities of the individual accessory proteins has generated distinct, multi-step models for the two biosynthetic pathways.
Section snippets
[NiFe] cluster
The dinuclear catalytic center of the [NiFe] enzyme, which is bound to the larger subunit of the dimeric protein, is composed of a nickel ion bound to four cysteine residues, two of which bridge to an iron ion that is also coordinated to one CO and two CN ligands (Figure 1) [5]. In Escherichia coli, which expresses at least three [NiFe] hydrogenases [3], most of the accessory proteins required for the biosynthesis of this metallocenter are encoded by the hyp (hydrogenase pleiotropic) genes
[FeFe] cluster
The active site metallocenter in the [FeFe] enzymes, called the H cluster (Figure 1), contains two iron ions bound to multiple CO and CN ligands and bridged by an organic cofactor proposed to be di(thiomethyl)amine or 1,3-propanedithiolate [4, 40]. One of these irons is ligated by a cysteine residue that also connects the catalytic site to a [4Fe–4S] cluster. Hyp homologs were not found in organisms expressing only [FeFe] hydrogenases, suggesting that this active site is assembled by distinct
Fe–S cluster
In addition to the biosynthesis of dinuclear catalytic clusters, [NiFe] and [FeFe] hydrogenases possess domains or separate subunits containing Fe–S clusters that are required for moving electrons from upstream donors or to downstream acceptors [3]. The production of the final enzyme requires the biosynthesis of these Fe–S clusters as well as, in some cases, processing of these subunits, association with the catalytic subunit and transfer through membranes [8]. A dedicated biosynthetic pathway
Conclusions
Despite the similar structures of the hydrogenase metallocenters, they are assembled by distinct accessory protein factors. However, there are some common themes in the biosynthetic pathways. The biosynthesis of the metallocenters for [NiFe]- and [FeFe]-hydrogenases both employ GTPases with similar postulated roles, as nickel metallochaperone for [NiFe]-hydrogenases and iron cluster transfer for [FeFe]-hydrogenases. Both [FeFe]- and [NiFe]-hydrogenases have iron centers with CN and CO ligands
Update
A recent analysis of Helicobacter pylori HypA by X-ray absorption spectroscopy revealed a dynamic coordination environment around the zinc ion [54]. In the presence of nickel the zinc is bound in a tetrathiolate coordination sphere, as predicted for the E. coli protein [19]. However, in the absence of nickel one of the cysteines is replaced by an N or O donor. This structural change around the zinc suggests that it has a role in nickel binding.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
This work was funded by grants from the Natural Sciences and Engineering Research Council of Canada and the Canada Research Chairs Program.
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