A protein machinery for [FeFe]-hydrogenase maturation
In order to be catalytically active after its synthesis, the [FeFe]-hydrogenase polypeptide encoded by the hydA gene, and thus named HydA in the following, has to incorporate the H-cluster and, when required, accessory [Fe–S] clusters. This post-translational process is extraordinarily complex as it involves a number of difficult reactions including: (i) the assembly of the accessory [Fe–S] clusters; (ii) the synthesis of CO, CN– and the adt2– bridging ligand; (iii) the assembly of the [2Fe]-subcluster or a close precursor; (iv) its incorporation into the enzyme already containing the [4Fe–4S]H component of the H-cluster. These reactions have to be tightly controlled because they produce the toxic CN– and CO molecules, as well as the hydrolytically sensitive adt2– ligand. This suggests that within the cell the synthesis of these ligands, their binding to Fe, and the transfer of organometallic intermediates occur via concerted processes, probably within a multiprotein complex consisting of the maturases and the hydrogenase target.
It is well established, using the [FeFe]-hydrogenase from C. reinhardtii, that the [4Fe– 4S]H component of the H-cluster is assembled prior to [2Fe]-subcluster insertion into the hydrogenase.10 Already in 1987 the expression of the two structural genes of [FeFe]-hydrogenase from Desulfovibrio vulgaris Hildenborough (DvH) in E. coli was reported to yield an inactive enzyme, which contained about 12 Fe atoms/hydrogenase molecule.11 At a time when the exact nature of the active metal center of [FeFe]-hydrogenases was unknown, only the two ferredoxin-like [4Fe–4S] clusters were supposed to have been assembled in the recombinant hydrogenase.
However, the reported number of Fe ions per protein molecule strongly suggests that the [4Fe–4S]H cluster was also assembled in the DvH hydrogenase. Thus the currently accepted model implies separate mechanisms, with a specific machinery being exclusively involved in the synthesis/assembly of the [2Fe]-subcluster and its transfer to an inactive [FeFe]-hydrogenase polypeptide containing all [Fe–S] clusters including the [4Fe–4S]H cluster. The assembly of these [Fe–S] clusters is likely to depend on the ISC and SUF machineries which are essential for maturation and activation of [Fe–S] enzymes in all prokaryotes.
The [FeFe]-hydrogenase maturation protein machinery, named HYD in the following, was initially discovered in the eukaryotic green algae C. reinhardtii, in 2004.13 The disruption of either the hydEF or hydG gene resulted in a mutant that proved to be unable to produce hydrogen, even though full-length hydrogenase accumulated. Furthermore, these genes and the two C. reinhardtii [FeFe]-hydrogenase genes, hydA1 and hydA2, were shown to be co-regulated. Finally, heterologous expression of an active C. reinhardtii HydA1 protein, CrHydA1, in E. coli absolutely required the co-expression of both hydEF and hydG genes. From all these results it could be concluded that HydEF and HydG provide the minimal protein machinery involved in the synthesis and assembly of the [2Fe]-subcluster of CrHydA1 active site. Whether other proteins are required for an optimal maturation process seems to be excluded so far.
Analysis of the hydEF gene from C. reinhardtii demonstrated that it contains two domains, which are homologous to two distinct genes coding for the proteins HydE and HydF, which are found exclusively in prokaryotic organisms naturally expressing a [FeFe]-hydrogenase. Thus the genomic organization of the three hyd genes, hydE, hydG, hydF, involved in [FeFe]-hydrogenase maturation, varies from one organism to another with three possible patterns; (i) independent genes scattered in the genome (C. acetobutylicum, C. perfringens); (ii) fused genes (C. reinhardtii); (iii) genes organized in operons (D. desulfuricans, B. thetaiotaomicron).
HydF, an iron-sulfur protein
Analysis of an alignment of HydF protein primary sequences from different [FeFe]-hydrogenase-containing microorganisms showed the complete conservation of three cysteine residues located in the C-terminal region of the protein, within a CxHxnHCxxC motif which was proposed to chelate the reconstituted cluster. Cendron L. and coworkers reported the X-ray structure (paragraph 184.108.40.206) of HydF from Thermotoga neapolitana (TnHydF) but unfortunately in its apoform where the Cys residues were involved in intramolecular and intermolecular S-S bridges.30 Those cysteines were found, by site-directed mutagenesis, to be essential for the assembly of the [4Fe–4S] center.31 Furthermore, mutation of the three cysteines into serine in HydF from C. acetobutylicum resulted in proteins inactive for C. acetobutylicum [FeFe]-hydrogenase maturation in an E. coli maturation system, demonstrating the cluster to be essential for HydF function.16,31 While the cysteines provide three ligands to the cluster, the ligand for the fourth Fe atom of the cluster has been a matter of controversy.
While it seemed to vary from one species to another, it has been well established by spectroscopic studies, in particular HYSCORE experiments, that the fourth coordination site of the cluster is readily exchangeable. Indeed, the cluster can bind exogenous imidazole,23,31 a histidine from the N-terminal His-tag of a tagged preparation of HydF,32 or a synthetic diiron complex mimicking the [2Fe]-subcluster of HydA, via a cyanide bridge.6 This has led, in the case of HydF from T. neapolitana, to the proposal that the exchangeable ligand is a solvent molecule (H2O or OH–), coupled to other water molecules, as suggested by ESEEM and HYSCORE spectroscopy.33,34 HYSCORE spectroscopy and site-directed mutagenesis also allowed excluding unambiguously a histidine coordination from the CxHxnHCxxC motif in HydF from T. maritima and T. neapolitana.23,31,32 In contrast, it seems that the HydF protein from C. acetobutylicum uses the C-terminal histidine H352 as a ligand of the cluster as shown by HYSCORE spectroscopy and site directed mutagenesis.31 On the other hand, the H352A mutant can still assemble a [4Fe–4S] cluster. All these observations suggest a weakly bound ligand, probably required for functional assembly of the transient [2Fe]-subcluster precursor in HydF. On the other hand, mutations of either histidine of the CxHxnHCxxC motif in HydF from C. acetobutylicum resulted in severe impairment of HydA maturation suggesting that these histidines, which are structurally close to the cluster, play an important role in the chemical processes associated either in the assembly of the diiron complex in HydF or its dissociation for transfer to apo-HydA, lacking the [2Fe]-subcluster.31,35 The impact of similar mutations in other HydFs on HydA activation has not been studied so that it is impossible to make a generalization of the role of these histidines so far.
HydF, a nucleotide-binding protein
Primary sequence analysis of the N-terminal domain of HydF from T. maritima and all homologous proteins from various [FeFe]-hydrogenase-containing microorganisms revealed the presence of several conserved consensus sequences, similar to those involved in guanine nucleotide binding in Small-G proteins. The first motif is the (G/A)X4GK(T/S) sequence responsible for the binding of- and-phosphate groups of the nucleotide (the P loop). Mutant HydF proteins from C. acetobutylicum in which either one of the glycines or the serine of the P-loop motif has been changed to alanine have been shown to be unable, in combination with HydE and HydG, to activate HydA from the same organism.16 Other remarkable features present in HydF proteins are: (i) a conserved threonine residue that might correspond to the residue of the G2 loop involved in Mg2+ binding; (ii) the DX2G sequence that represents the G3 loop involved in the interaction with the-phosphate and Mg2+, and (iii) the G4 loop, (N/T)(K/Q)XD, which is supposed to interact with the nucleotide part of GTP. GTP binding to HydF was indeed demonstrated using fluorescence spectroscopy, with a Kd value of 3 µM for the dissociation of the HydF protein-GTP complex, and furthermore HydF was shown to catalyze GTP hydrolysis to GDP in the presence of Mg2+.23 A GTPase activity has also been observed in the case of HydF from Clostridium acetobutylicum.26,27 This activity is affected by the nature of the monovalent cation in the reaction buffer with the highest activity obtained with K+ and Rb+, suggesting that a binding pocket for K+ exists near the active site. Furthermore, while it is not affected by the presence of HydA, it is stimulated by the presence of HydE and HydG. These results, together with the observed lack of effect of GTP on the activation of HydA by HydF, suggest that the GTPase activity of HydF plays no role in the transfer of the transient diiron complex from HydF to HydA but more likely contributes to assembling this complex on HydF, in concert with HydE and HydG activity. Finally, it was shown also that the GTPase activity was independent on the presence of the cluster.
It is interesting to note that HypB, a protein with GTPase activity and Kd values for GTP comparable to those displayed by the HydF protein, is involved in the maturation of [NiFe]-hydrogenase.38,39 It has been shown that HypB participates in the GTP-dependent insertion of the nickel atom into an hydrogenase form already containing the Fe(CO)(CN)2 motif. It seems to be a common theme that nucleotidase activity is required in metal-containing active site biosynthesis.
In addition to the HydF and HypB proteins, one can mention the CooC38,40 and UreG41 proteins, involved in nickel insertion during maturation of carbon monoxide dehydrogenase and urease, respectively, and NifH, which functions in the ATP-dependent insertion of the FeMoco into apo-nitrogenase.
HydG catalyzes tyrosine conversion to CO and CN
In contrast to HydE, detailed knowledge of HydG structure and function has resulted from exquisite biochemical, spectroscopic and structural studies in the last few years. It has been very early established that HydG proteins are “Radical-SAM” enzymes which contain two [4Fe–4S] clusters, both absolutely required for activity.24 The first one, [4Fe–4S]RS, is chelated by the characteristic “Radical-SAM” CysX3CysX2Cys motif while the second, [4Fe– 4S]AUX, uses the cysteines from another conserved C-terminal CysX2CysX22Cys motif. The six cysteines were shown by site-directed mutagenesis to be essential for activity.16 Thus both clusters have a free coordination site, the first one for binding SAM and the second for binding a key mononuclear Fe complex, as discussed below.
An amino acid sequence comparison of HydG with other members of the “Radical-SAM” protein family indicated that tyrosine lyases (ThiH) were among the most closely related to HydG with 27% identity. 35% of the identities between HydG and ThiH correspond to strictly conserved residues in all the known amino acid sequences of both families. ThiH catalyzes the conversion of tyrosine into para-cresol and dehydroglycine (DHG), a precursor in thiazole biosynthesis.50 This genomic analysis and the remarkable homology between ThiH and HydG led to discover that tyrosine is the substrate of HydG.51 This result extends the sequence homology of HydG and ThiH to a remarkable functional similarity, which provided a solid basis for subsequent mechanistic investigations.
P. Roach and coworkers then showed that CO and CN– ligands are produced from SAM-dependent radical cleavage and decomposition of tyrosine.52,53 Cyanide was detected and quantified after derivatization with a fluorescent probe then analyzed by HPLC and LCMS.
Redox states of the H-cluster of [FeFe]-hydrogenases
As described above, the H-cluster of [FeFe]-hydrogenases is composed of a [4Fe–4S] cluster, linked to a [2Fe]-subcluster through a cysteine bridge. Each Fe atom of the binuclear part has one CO and one CN– ligand and connects to the other Fe through an additional bridging CO ligand and an azadithiolate ligand. The two irons are called proximal (Fep) and distal (Fed) respectively. Both the cubane and the binuclear part of the H-cluster are redox active and during catalysis they can change their oxidation states. In particular the cubane has access to the redox states [4Fe–4S]+2 and [4Fe–4S]+ while the [2Fe]-subcluster can have different configurations, Fep(II)Fed(II), Fep(I)Fed(II) and Fep(I)Fed(I). Some of them are paramagnetic (shown in bold) and observable by EPR techniques.
Up to now only two [FeFe]-hydrogenases, HydA from Desulfovibrio desulfuricans (DdH) and from C. reinhardtii (CrHydA1), have been thoroughly studied for their redox behavior and a combination of FTIR, EPR and Mössbauer techniques allowed the isolation and identification of various states of the H-cluster, which differ from each other substantially.
CO and CN ligands give rise to FTIR stretches in a zone free of other protein bands and their position is sensitive to the charge density on the binuclear core and can be used to observe the various H-cluster states.64 In particular, the reduced states present lower frequencies of stretches due to the higher component of back-bonding. Moreover, the paramagnetic states of the H-cluster produce very characteristic EPR spectra facilitating their identification. Not all the redox states are observable in all species. In the particular case of the aerobically purified DdH HydA, the H-cluster is found in the inactive air-stable Hoxair state, where the cubane is in the [4Fe–4S]2+ state and the binuclear in the Fe(II)Fe(II) state, thus EPR silent.
Mild reduction conditions lead to an intermediate redox state called Htrans, where the electron is stored on the cubane giving rise to the [4Fe–4S]+-Fe(II)Fe(II), which shows the typical spectrum of reduced [4Fe–4S]+ cluster.65 This redox state is isoelectronic with the Hox state, [4Fe–4S]2+-Fe(I)Fe(II), which has peculiar FTIR and EPR signatures. The FTIR spectra of Hox of various [FeFe]-hydrogenases present a characteristic signal for the bridging CO around 1800 cm-1.3 The diiron subcluster, Fe(I)Fe(II), is paramagnetic (S=1/2) with similar g values (g= [2.10, 2.04, 2.00]) for all [FeFe]-hydrogenases while the [4Fe–4S]H is oxidized (2+) and thus EPR silent. Upon inhibition by external CO gas, a new state is formed with the same electronic structure of Hox, where a CO molecule binds the free coordination site on the 2Fe-subcluster. This Hox-CO inhibited state gives rise to significant changes in the FTIR and EPR spectra. The EPR (S=1/2) signal is axial and is often observed in samples exposed to light and/or oxygen, where the CO ligands dissociated from the destroyed H-cluster are captured by active sites still intact (cannibalization).66 While the Hoxair and Htrans are not observed in any other [FeFe]-hydrogenase, the Hox state and its CO inhibited version Hox-CO are found in all [FeFe]-hydrogenases.
Upon further reduction of the Hox state, the Hred state is formed. Here, the cubane is in the [4Fe–4S]2+ state and the binuclear in the Fe(I)Fe(I) and thus both are EPR silent. The FTIR stretches shift to lower wavenumbers confirming the reduction of Fe ions in the [2Fe]-subcluster.
Table of contents :
Abbreviations and Acronyms
Chapter I: Introduction
1.1 Metalloprotein: metal ions in living systems
1.2 Iron-sulfur cluster proteins
1.3 Biosynthesis of Fe–S clusters
1.4 Hydrogenase enzymes
1.5 The metal center of [FeFe]-hydrogenases
1.5.1 A protein machinery for [FeFe]-hydrogenase maturation
1.5.2 The Hyd proteins
1.5.3 The HydF protein
220.127.116.11 HydF, a scaffold protein
18.104.22.168 HydF, an iron-sulfur protein
22.214.171.124 HydF, a nucleotide-binding protein
126.96.36.199 Structure of the HydF protein
1.5.4 The HydE protein
188.8.131.52 HydE, a radical-SAM enzyme
184.108.40.206 HydE: a second, non-essential, cluster
220.127.116.11 HydE partners
18.104.22.168 Structure of the HydE protein
1.5.5 The HydG protein
22.214.171.124 HydG catalyzes tyrosine conversion to CO and CN
126.96.36.199 HydG: a radical mechanism
188.8.131.52 HydG: an unprecedented [5Fe-4S] cluster
184.108.40.206 Structure of the HydG protein
220.127.116.11 HydG enzyme mechanism
1.5.6 Mechanism of maturation of [FeFe]-hydrogenases
1.5.7 Redox states of the H-cluster of [FeFe]-hydrogenases
1.5.8 Maturase-free Chemical maturation: a unique technological tool
1.6 Artificial hydrogenases
1.6.1 Artificial hydrogenases based on synthetic diiron complexes
1.6.2 Ni-based artificial hydrogenases
1.6.3 Artificial hydrogenases based on synthetic cobalt complexes
Aim of the project
Results and discussion
Chapter II: The [FeFe]-hydrogenase from Megasphaera elsdenii
2.1 Expression and aerobic purification of MeHydA
2.2 [Fe–S] cluster reconstitution of apo-MeHydA
2.3 Static light scattering of apo-MeHydA
2.4 EPR spectroscopic characterization of the iron-sulfur MeHydA
2.5 Chemical maturation
2.5.1 Incorporation of synthetic [Fe2(adt)(CO)4(CN)2]2– complex
2.5.2 Incorporation of synthetic [Fe2(pdt)(CO)4(CN)2]2– complex
2.6 FTIR and EPR spectroscopic characterization of holo-MeHydA
2.7 Conclusion and future perspectives: biotechnological application?
Chapter III: A truncated form of M. elsdenii [FeFe]-hydrogenase
3.1 Strategy 1: Cys to Ser mutations
3.1.1 Expression and purification of CystoSerMeHydA construct
3.2 Strategy 2: MeH-HydA
3.2.1 Cloning experiment
3.2.2 Expression and aerobic purification of apo-MeH-HydA
3.2.3 [4Fe–4S]H cluster reconstitution of MeH-HydA apoprotein
3.2.4 Spectroscopic characterization of FeS-MeH-HydA
3.2.5 Chemical maturation of FeS-MeH-HydA with [Fe2(adt)(CO)4(CN)2]2- synthetic complex
18.104.22.168 MeH-HydA: new maturation conditions led to higher maturation
3.2.6 FTIR and EPR characterization of chemically maturated MeH-HydA
3.2.7 Conclusion and future perspectives
Chapter IV: The [FeFe]-hydrogenase maturase HydF
4.1 HydF protein from Thermotoga maritima
4.1.1 Expression, purification and iron-sulfur reconstitution of HydF from
4.1.2 Insertion of synthetic complexes 1 and 2 onto TmHydF: hydrogenase activity
4.1.3 X-ray structure of apo-TmHydF, the apoform of HydF from Thermotoga maritima
4.2 Preparation and characterization of iron-sulfur reconstituted HydF from
Thermosipho melanesiensis and Clostridium Thermocellum
4.2.1 X-ray structure of TmeHydF: a [4Fe–4S] with an unexpected and exchangeable ligand
4.2.2 Preparation and characterization of 1- and 2-TmeHydF hybrids
4.2.3 Hydrogenase activity of 2-TmeHydF hybrid protein
4.2.4 Site-directed mutagenesis of TmeHydF: E305C and E305H
4.3 Conclusion and future perspectives
Chapter V: Materials and Methods
5.1 Biologic material
5.1.1 Competent cells
5.1.3 Growth media
5.1.4 Molecular Biology
22.214.171.124 Plasmid preparation
5.2 Biochemical methods: Protein expression
5.2.1 TmHydF: HydF from Thermotoga maritima
5.2.2 HydF from Thermosipho melanesiensis (TmeHydF) and Clostridium thermocellum (CtHydF)
126.96.36.199 E305C and E305H TmeHydF mutants
5.2.3 MeHydA: the [FeFe]-hydrogenase from Megasphaera elsdenii
5.2.4 MeH-HydA: the [FeFe]-hydrogenase truncated form of Megasphaera elsdenii
5.2.5 CsdA: the Cysteine Desulfurase from E. coli
5.3 Proteins purification
5.3.1 TmHydF: HydF from Thermotoga maritima
5.3.2 HydF from Thermosipho melanesiensis (TmeHydF) and Clostridium thermocellum (CtHydF)
188.8.131.52 E305C and E305H TmeHydF mutants
5.3.3 CsdA from E. coli.
5.3.4 MeHydA: the [FeFe]-hydrogenase from Megasphaera elsdenii
5.3.5 MeH-HydA: the [FeFe]-hydrogenase truncated form of Megasphaera elsdenii
5.4 Biochemical methods: [Fe–S] cluster reconstitution of apo-proteins
5.4.1 Iron-sulfur reconstitution with 57Fe
5.4.2 [Fe–S] protein preparation for EPR and HYSCORE measurements
184.108.40.206 TmeHydF, E305C and E305H mutants
220.127.116.11 MeHydA and MeH-HydA
5.5 Chemical methods: Insertion of [Fe2(adt/pdt)(CO)4(CN)2]2- onto HydF and HydA
5.5.1 Synthesis of (Et4N)2[Fe2(adt/pdt)(CO)4(CN)2] complexes
5.5.2 Insertion of [Fe2(adt/pdt)(CO)4(CN)2]2- complex onto MeHydA
18.104.22.168 Hox state
22.214.171.124 Hox-CO inhibited state
126.96.36.199 Hred state
5.5.3 Insertion of [Fe2(adt)(CO)4(CN)2]2- complex onto MeH-HydA
188.8.131.52 Hox, Hox-CO and Hred states
5.5.4 Insertion of [Fe2(adt/pdt)(CO)4(CN)2]2- complex onto HydF proteins
5.6 Hydrogenase-like activity
5.6.1 Calibration curve for H2 quantitation
5.6.2 H2 detection via Gas Chromatography of MeHydA and MeH-HydA proteins
5.6.3 H2 detection via a miniaturized Clark-type hydrogen sensor
184.108.40.206 Methyl viologen assay for TmHydF, TmeHydF and MeHydA proteins
220.127.116.11 Photocatalytic H2 production assay driven by Ru(bpy)3 2+
5.7 Determination of protein concentration
5.8 Determination of cofactor concentration
5.8.1 Iron quantitation: Fish method
5.8.2 Sulfur quantitation: Beinert
5.9 Spectroscopic characterization
5.9.1 Uv-Visible spectroscopy
5.9.2 EPR spectroscopy
5.9.3 HYSCORE spectroscopy
5.9.4 FTIR spectroscopy
5.9.5 Mössbauer spectroscopy
5.9.6 X-ray Crystallography
18.104.22.168 Refinement statistics