The crystal structure of the escherichia coli o2sensitive c19g nife hydrogenase 1 variant shows that the mutation results in a novel fes cluster, proximal to the nife active site. While the proximal cluster of the native o2tolerant enzyme can transfer two electrons to that site, epr spectroscopy shows that the modified cluster can transfer. Novel roles for fe s clusters in stabilizing or generating radical intermediates article pdf available in pure and applied chemistry 704. Examples include both the 8fe and the 7fe clusters in nitrogenase. Due to the absence of additional fes clusters as found in bacterial fefe hydrogenases, it was possible to specifically study its catalytic iron. The crystal structure of an oxygentolerant hydrogenase. Delivery of ironsulfur clusters to the hydrogenoxidizing nife. Because there is only a single fes cluster binding motif in hydf, it is tempting to assume that the 2fe unit is directly bound to the 4fe4s cluster. Biochemical analysis of the interactions between the. Compartmentalisation of fefehydrogenase maturation in. An investigation of hydrogenase i and hydrogenase ii from clostridium pasteurianum by resonance raman spectroscopy.
The fefe hydrogenase active site consists of the h. Novel, oxygeninsensitive group 5 nifehydrogenase in. Characterization of a unique fes cluster in the electron transfer. Xray structural, functional and computational studies of. The medial 3fe4s3cys cluster and the distal 4fe4s3cys1his cluster are well conserved among the nife hydrogenases. We also investigated whether the presence of a fes cluster precursor on the hydf eg scaffold is required for its interaction with the hydrogenase. Unraveling the role of the protein environment for fefe.
Discovery of novel fefehydrogenases for biocatalytic h2. In this study, we develop and apply a novel combination of allatom molecular dynamics and coarsegrained cg analysis to characterize two important steps of the catalytic cycle of fefe hydrogenase. Here we report the assembly of the ironsulphur framework of the active site of irononly hydrogenase the h cluster, and show that it functions as an electrocatalyst for proton reduction. Pdf an investigation of hydrogenase i and hydrogenase ii. Advanced paramagnetic resonance studies of the feonly. The fefe hydrogenase crhyda1 of the green alga chlamydomonas reinhardtii is the smallest hydrogenase known and can be considered as a minimal unit for biological h 2 production. Delivery of ironsulfur clusters to the hydrogenoxidizing nifehydrogenases in escherichia coli requires the atype carrier proteins erpa. The fefehydrogenase active site cluster is termed the h cluster, and exists. The accessory fes cluster binding domains f clusters are typically located at the n terminus of the h cluster binding domain and are either. Fefe and nifehydrogenase diversity, mechanism, and. This socalled group 5 nife hydrogenase is widespread in soilliving actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward h2. The largest structural differences among fefehydrogenases are related to their respective complement of accessory fes clusters f cluster, which putatively function to regulate electron transfer to andor from the h cluster active site. Indeed, the fes s cluster, or at least its fifth iron, apparently has not been readily reconstituted in most of the in vitro experiments reported to date.
Pinching maness, national renewable energy laboratory. The unusual 4fe3s cluster proximal to the active site plays a crucial role in allowing a class of nifehydrogenases to function in the presence of o2 through its unique ability to undergo two rapid, consecutive oneelectron transfers. Electrons are transferred over long distances along chains of fes clusters in hydrogenases, mitochondrial complexes, and many other respiratory enzymes. The h cluster and a small number of other complex fes clusters. While the proximal cluster of the native o 2tolerant enzyme can transfer two electrons to that site, epr spectroscopy shows that the modified cluster can transfer only one electron, this shortfall. Nife hydrogenase is a type of hydrogenase, which is an oxidative enzyme that reversibly convert molecular hydrogen in prokaryotes including bacteria and archaea. Ironsulfur clusters of hydrogenase i and hydrogenase ii of.
This indicates that the hyda1 fes cluster can only be built by the chloroplastic fes cluster. In addition, an unusual epr signal has been attributed to a novel 6fe cluster that was proposed to be the active site and was called the h cluster 5. Synthesis of the hcluster framework of irononly hydrogenase. In this study, we develop and apply a novel combination of allatom molecular dynamics and coarsegrained cg analysis to characterize two important steps of. Fefeahydrogenase maturation in chlamydomonas reinhardtii. The structure of this 6fe cluster has been determined by xray crystallographic analysis2,3and comprises a4fe4ssubcluster, produced by canonical fes cluster.
The ironsulfur clusterfree hydrogenase hmd is a metalloenzyme. The purified proteins consist of two different subunits with molecular masses of approximately 60 and 40 kda and contain a redox. Based on spectroelectrochemical studies, this enzyme exhibits. Recently, a novel group of nifehydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. Get a printable copy pdf file of the complete article 1. Characterization of a unique fes cluster in the electron transfer chain of the oxygen tolerant nife hydrogenase from. Duderstadt department of chemistry and center for metalloenzyme studies, university of georgia, athens, ga. Using site saturation mutagenesis, a random approach that is a novel technique in fefe hydrogenase field, we demonstrated that aspartic acid is the only residue that can. A novel fes cluster in feonly hydrogenases request pdf. The h cluster exists as a 4fe4s cluster connected via a singlebridging cysteine thiolate to a binuclear 2fe center having biologically unique diatomic co and cn. Ironsulfur clusters are versatile electron transfer cofactors, ubiquitous in. The metalsulphur active sites of hydrogenases catalyse hydrogen evolution or uptake at rapid rates. Catalysis occurs at the h cluster, a carbon monoxide, cyanide, and dithiomethylaminecoordinated 2fe subcluster bridged via a cysteine to a 4fe4s cluster. Hydrogenase enzymes are natural biocatalysts that might be harnessed to reduce the cost of hydrogen gas production.
To this end, we obtained a new recombinant hydf6his protein in which one of the three highly conserved cysteine residues belonging to the fes cluster binding consensus sequence i. Carbon monoxide dehydrogenase and the fefe hydrogenase also feature unusual fes clusters. It is usually presumed that electron transfer is fast in these systems, despite the fact that there has been no direct measurement of rates of fes to fes electron transfer in any respiratory enzyme. Fe hydrogenase ironsulfurclusterfree hydrogenase catalyzes the reversible reduction of methenyltetrahydromethanopterin. Nicolet y, piras c, legrand p, hatchikian ce, fontecillacamps jc. Hydrogenases from methanogenic archaea, nickel, a novel cofactor, and h2 storage. Hydrogen from water in a novel recombinant oxygentolerant cyanobacterial system philip d. Advanced paramagnetic resonance studies of the feonly hydrogenase i from clostridium pasteurianum cpi joshua telser 1,2, paul m. The cartoon depicts various ferredoxinlike fes clusterbinding domains, gas diffusion pathways, and a. The catalytic site on the enzyme provides simple hydrogenmetabolizing microorganisms a redox mechanism by which to store and utilize energy via the reaction shown in figure 1.
Hoffman 2 1chemistry program, roosevelt university, chicago, il 60605 usa, 2 department of chemistry, northwestern university, evanston, il 60208 usa, and 3 department of chemistry and. This socalled group 5 nifehydrogenase is widespread in soilliving actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward h 2. In addition to these ligands, the irons of the subcluster are bridged by a non. The accessory fes cluster binding domains f clusters are typically located at the n terminus of the h cluster binding domain and are either 4fe4s or. These latter factors are however less likely to influence the outcome of the screening as the motifs required for a gene to encode for an fefe hydrogenase are well defined 1923 and the e.
Biosynthesis of this unique metallocofactor is accomplished by three maturase enzymes denoted hyde, hydf, and hydg. Figure 1 a amino acid sequences of desulfovibrio desulfuricans fe hydrogenase ddh and clostridium pasteurianum fe hydrogenase i cpi, deduced from the gene sequences, aligned to take into account the threedimensional structure homology. Despite intensive efforts, however, the understanding of how hydrogenases react with h2 is only in its infancy. The crystallographic characterization of feonly hydrogenases 1,2 has revealed a striking resemblance of the diiron subsite of the h cluster to known fe 2 sr 2 co 6 r organic group. This property helps to neutralize reactive oxygen species. On the novel h2activating ironsulfur center of the feonly hydrogenases. Biosynthesis of this unique metallocofactor is accomplished by three maturase enzymes. In the oxygentolerant hydrogenase i from aquifex aeolicus such electron wires form a relay to a diheme cyt b, an integral part of a respiration pathway for the reduction of o2 to water. A special 6 cysteinecoordinated fe 4 s 3 cluster was found in oxygentolerant membranebound nife hydrogenases.
Cys and his residues are implicated in 4fe4s and 2fe2s cluster coordination. Structural comparisons a novel fes cluster in feonly. Ironsulfur cluster states of the hydrogenase maturase hydf. Colocalization of hydg, hyde, and hydf with fe hydrogenase in the hydrogenosomes strongly supports our hypothesis that these three proteins are required for the synthesis of active fe hydrogenase, as demonstrated for c. The fefehydrogenase maturation protein hydf contains a. Characterization of a unique fes cluster in the electron. Here the role of c298 in fefe hydrogenase activity is investigated because of its proximity to the active site and its putative role in proton transfer to the h cluster.
Structural biochemistryenzymefemo cofactor and fefe. Stripp b and gustav berggren a a new screening method for fefehydrogenases is described, circumventing the need for specialized. Nature utilizes fefe hydrogenase enzymes to catalyze the interconversion between h2 and protons and electrons. Hydrogenase h cluster hydrogenase h cluster is composed of a diiron cluster with unusual nonprotein ligands bridges to a 4fe4s cluster. Novel roles for fes clusters in stabilizing or generating. The two enzymes also contain ferredoxintype fouriron centers or f clusters.
The exchange activities of fe hydrogenase ironsulfurclusterfree. A novel fes cluster in feonly hydrogenases sciencedirect. Epr spectroscopic studies of the fes clusters in the o2. In the 4fe4s subcluster, one of the coordinating cysteine thiolates bridge between 4fe4s subcluster and the 2fe subcluster, which each fe ion in the cluster is coordinated by terminal carbon monoxide. Insights into fefehydrogenase structure, mechanism, and.
Nife hydrogenases catalyze the reversible reduction of molecular hydrogen to protons. In the oxygentolerant hydrogenase i from aquifex aeolicus such. Site saturation mutagenesis demonstrates a central role. Mechanistic details and the role of the medial and distal clusters remain unresolved. Discovery of novel fefehydrogenases for biocatalytic h. Rates of intra and intermolecular electron transfers in. Proceedings of the national academy of sciences 107 23, 1044810453, 2010. Ironsulfur cluster coordination in hydf was investigated by site. Novel reactions involved in energy conservation by. The cpi and ddh structures revealed the unique active site metal cluster, termed the h cluster, at which catalysis takes place.
Amino acid sequence comparison with oxygensensitive hydrogenases showed. Nhe, which was probed by electrochemistry and gas chromatography. Pdf novel roles for fes clusters in stabilizing or. New insights into hydrogenase maturation article pdf available in jbic journal of biological inorganic chemistry 124.
Nicolet y, lemon bj, fontecillacamps jc, peters jw. Novel roles for fe s clusters in stabilizing or generating radical intermediates michael k. The positions of the f and h clusters in the respective f and hdomains of c. Author links open overlay panel yvain nicolet a brian j lemon b juan c fontecillacamps a john w peters b. Ironsulfur clusters are versatile electron transfer cofactors, ubiquitous in metalloenzymes such as hydrogenases. Ddh is a dimeric periplasmic protein of 53 kda 43 kda and 10 kda for the large and.
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