The purple sulphur phototrophic bacterium, Thiocapsa roseopersicina BBS, contains several NiFe hydrogenases. One of these enzymes (HynSL) is membrane associated, remarkably stable and can be used for practical applications. HupSL is also located in the photosynthetic membrane, its properties are similar to other known Hup-type NiFe hydrogenases. A third hydrogenase activity was located in the soluble fraction and was analogous to the NAD-reducing hydrogenases of cyanobacteria. The hoxEFUYH genes are transcribed together. HoxE is needed for the in vivo electron flow to and from the soluble hydrogenase. Some of the accessory genes were identified using random mutagenesis, and sequencing of the T. roseopersicina genome is in progress. The HupD, HynD and HoxW gene products corresponded to the proteases processing the C-termini of the three NiFe hydrogenases respectively. HypF and HupK mutants displayed significant in vivo H2 evolution, which could be linked to the nitrogenase activity for the ΔhypF and to the bidirectional Hox activity in the ΔhupK strain. Both HypC proteins are needed for the biosynthesis of each NiFe hydrogenase. The hydrogenase expression is regulated at the transcriptional level through distinct mechanisms. The expression of hynSL is up-regulated under anaerobic conditions with the participation of an FNR (fumarate and nitrate reduction regulator)-type protein, FnrT. Although the genes encoding a typical H2 sensor (hupUV) and a two-component regulator (hupR and hupT) are present in T. roseopersicina, the system is cryptic in the wild-type BBS strain. The hupR gene was identified in the gene cluster downstream from hupSL. Introduction of actively expressed hupT repressed the hupSL gene expression as expected by analogy with other bacteria.
Understanding the molecular fundamentals of hydrogen production and utilization in biological systems is a goal of supreme importance for basic and applied research . The key enzyme in biological H2 metabolism is hydrogenase. It should be noted that hydrogenases can help us in two ways in practical applications: they may catalyse both H2 generation (e.g. photobiological or fermentative) and H2 oxidation (e.g. in fuel cells).
In metal-containing biological catalysts, it is the protein matrix, surrounding the metal centres, which provides the unique environment for the Fe and Ni atoms and allows hydrogenases to function properly, selectively and effectively. Hydrogenases are ancient enzymes, hence their protein sequence and structure is rather conserved. The NiFe hydrogenases are composed of at least two distinct polypeptides, containing highly conserved metal-binding domains. The large subunit harbours the NiFe active centre, fastened to the protein by four cysteine ligands. Similar heterobinuclear NiFe centres are not known in any other metalloenzyme. The small subunit contains 2–3 Fe4S4 clusters, which are precisely and equally spaced, 15 Å (1 Å=0.1 nm) apart, and thus, form a conducting wire inside the protein to facilitate the transport of electrons between the active centre and the protein surface .
To develop suitable biocatalysts for future biotechnological applications, the structure–function relationship, biosynthesis and assembly of hydrogenases must be understood. A number of other gene-products govern the metal uptake, their attachment into the right place at the right time, formation of the unique CN and CO ligands, and the incorporation and fixation of this labile inorganic structure into the protein. Our present understanding suggests that the concerted action of, at least, 7–15 such accessory proteins is necessary for the formation of an active NiFe hydrogenase .
Hydrogenases in Thiocapsa roseopersicina
T. roseopersicina is a phototrophic purple sulphur bacterium, the strain marked BBS has been isolated from the cold water of the North Sea. It contains bacteriochlorophyll a and spirilloxanthin synthesized by Bch and Crt proteins . Its anaerobic photosynthesis uses reduced sulphur compounds (sulphide, thiosulphide or elementary sulphur), but it can also grow on organic compounds (sugar and acetate) in the dark. The bacterium possesses nitrogenase activity, and atmospheric N2 fixing is accompanied by H2 production .
Previous studies in our laboratory have revealed that T. roseopersicina contains at least two membrane-associated NiFe hydrogenases with remarkable similarities and differences. One of them (HynSL, for recent nomenclature change see ) shows extraordinary stability: it is much more active at 80°C, than approx. 25–28°C. Note that T. roseopersicina cannot grow above 30°C. HynSL of T. roseopersicina is also reasonably resistant to oxygen inactivation and stays active after removal from the membrane. The other NiFe hydrogenase, HupSL, is very sensitive to several environmental factors when separated from the photosynthetic membrane, thus it resembles the membrane-bound NiFe hydrogenases known from other microorganisms. The structural genes coding for these enzymes have been cloned and sequenced [5,6]. An additional NiFe hydrogenase activity was discovered in the cytoplasm of the bacterium. The cyanobacterial type NAD+-reducing HoxEFUYH is a pentameric, bidirectional NiFe hydrogenase . It produces H2 under nitrogenase-repressed conditions and it recycles the hydrogen produced by the nitrogenase in cells fixing N2. The five genes were localized on a single transcript. HoxE has a hydrogenase-related role, it probably participates in the electron transfer processes in vivo, but has no effect on the in vitro hydrogenase activity. This is the first example of the presence of a Hox-type hydrogenase in a phototrophic non-cyanobacterium. The genes of the putative regulatory hydrogenase, hupUV, have been identified, but the H2 sensing system is non-functional in this bacterium.
Thus, according to our current understanding, there are four distinct sets of genes coding for NiFe hydrogenases in T. roseopersicina, representing several NiFe hydrogenase forms thus far described in various microorganisms in a single cell. This makes T. roseopersicina one of the best candidates for studies of NiFe hydrogenase structure–function relationships and assembly .
From the fragmented information available, there is no clear answer as to why T. roseopersicina needs so many distinct hydrogenases. Our working hypothesis links this abundance of various NiFe hydrogenases to the fact that this bacterium should be capable of performing various metabolic activities (photoautotrophic, photoheterotrophic and heterotrophic metabolism) to survive in its natural habitat . Having numerous hydrogenases at hand increases the chances of survival for the bacterium and increases our chances to understand basic phenomena of hydrogenase catalysis.
The advantages of having at least four types of NiFe hydrogenases in T. roseopersicina are counter-balanced by the fact that most of the accessory genes needed for their assembly and biosynthesis are scattered in the genome. The downstream region of the hupSL structural gene cluster contains some of these genes: hupCDHI have been identified on the basis of their sequence homology to the corresponding auxiliary genes in other microorganisms . No pleiotropic accessory genes have been found around the hydrogenase coding structural gene clusters. Since some of the accessory genes are pleiotropic, the number of missing genes is estimated to be between 20 and 30. Most of these genes and their protein products are needed for the macromolecular structure–function studies; therefore, a systematic search has been launched using two approaches. We have begun sequencing the genome of T. roseopersicina, which will produce the complete sequence information of this bacterium. Random mutagenesis and screening for altered hydrogenase phenotypes allows us to identify those genes that play a significant role in the formation of the functionally intact enzymes. This is a straightforward approach if there is a good method available to screen the mutants and the mutation causes phenotypic change(s). To examine the specificity of the auxiliary proteins, hydrogenase deletion mutant strains were generated, and the effect of the mutation in the accessory genes was studied through hydrogenase activity assay measurements.
Mini Tn5 transposon mutagenesis was used to identify the hydrogenase accessory genes required for the maturation of the NiFe hydrogenase enzymes. In three mutants, the transposon was inserted into the hypD or the hypE gene abolishing all hydrogenase activities in the cells. The corresponding gene products have obviously fundamental roles in the formation of any NiFe hydrogenase in T. roseopersicina. Inactivation of NiFe hydrogenase biosynthesis in the hypF deficient mutant resulted in a 60-fold increase in hydrogen evolution capacity of T. roseopersicina under nitrogen fixing conditions . A detailed molecular investigation of the other mutant strains resulted in the identification of one locus containing the hupK-hypC1DE accessory genes and another one, where the hypC2, hynD genes were found [11,12].
The physiological functions of the HupK and HypC1, HypC2 proteins were further investigated. Reverse transcriptase–PCR and complementation experiments clearly indicated that the hypC1DE genes had their own regulatory element, independent from that of the hupK, but they could also be transcribed from the promoter of the hupK gene. In T. roseopersicina, the activities of both membrane-associated NiFe hydrogenases (HynSL and HupSL) decreased significantly in the absence of the HupK protein, whereas the soluble HoxEFUYH enzyme remained apparently unaffected . The inactivation of the hupK gene resulted in a nitrogenase-independent phototrophic H2 production. This protein does not occur in all microbes containing NiFe hydrogenase, hence the role of HupK is still uncertain. A protein–protein interaction was detected between HupK and a putative GroEL . Most probably, the putative GroEL is involved in the folding of HupK, however, an important and specific role for a GroEL homologue protein in hydrogenase metallocentre assembly cannot be excluded in T. roseopersicina. HupK resembles the large subunit of the NiFe hydrogenases, therefore it has been suggested to function as a scaffolding protein during metal cofactor assembly .
HypC is a small, chaperone-like protein participating in two protein complexes during hydrogenase maturation and, thus, a dual function has been assigned to it. HypC interacted with the large subunit of the hydrogenase 3 (HycE)  and it was shown to form a complex with the HypD protein  in Escherichia coli. The molecular phenotype of HypC mutations is strikingly different in T. roseopersicina  and in E. coli . In T. roseopersicina, the two HypC proteins are important for the maturation of all three hydrogenases even if they can partially substitute each other. Consequently, both HypC are truly pleiotropic accessory proteins in this bacterium. An intermediate protein complex formed during the maturation process of the HynL hydrogenase large subunit (HynL-HypC2) .
The hupD, hynD and hoxW genes of T. roseopersicina encode the C-terminal endoproteases of the corresponding NiFe hydrogenases. Hydrogenase-specific proteases have a function in one of the last steps of hydrogenase maturation, when the C-terminal end of the precursor large subunit polypeptide is cleaved, as soon as the NiFe heterobinuclear centre with its diatomic ligands has been successfully assembled and inserted into the active site of the enzyme [1,3]. Each endoprotease is highly specific for its hydrogenase and cannot replace each other in this function.
Regulation of biosynthesis
The diverse physiological functions of the various hydrogenases in T. roseopersicina imply distinct regulation mechanisms. The transcriptional control of hupSL expression is dependent on σ54, which binds to the conserved –24/–12 promoter element. A H2-driven signalling pathway, composed of a response regulator (HupR), a histidine protein kinase (HupT) and a cytoplasmic NiFe hydrogenase-like protein, named regulatory hydrogenase (HupUV) mediates the environmental signal for the transcriptional regulation machinery in Ralstonia eutropha and Rhodobacter capsulatus [17,18]. The elements of a similar system have been identified in T. roseopersicina, but H2-dependent expression of HupSL was not observed. HupR seems to be functional as mutation of its gene abolishes the hupSL transcription. The regulatory system appears to be cryptic, although the repressor function of HupT can be restored when expressing it using a T. roseopersicina promoter on a plasmid.
The hynSL expression is HupR independent and the transcription is up-regulated under anaerobic conditions. It has been demonstrated recently that anaerobic regulation depends on an upstream cis element of hynS. Two elements resembling FNR (fumarate and nitrate reduction regulator) binding sites have been recognized in this region. In vitro electrophoretic mobility shift and DNase I footprinting experiments verified the existence of two FNR-binding sites. The fnrT gene was identified, sequenced and knocked out to demonstrate the requirement of FnrT for the anaerobic induction of hyn expression. Interestingly, binding to the upstream DNA site for FNR required the presence of a functional downstream binding site. A putative −10 hexameric promoter element, usually dependent on σ70, was identified by in vitro transcription experiments upstream from the FnrT-regulated transcription-initiation start site.
International Hydrogenases Conference 2004: Independent Meeting held at the University of Reading, 24–29 August 2004. Edited by R. Cammack (King's College London, U.K.) and F. Sargent (University of East Anglia, Norwich, U.K.). Organized by R. Cammack and R. Robson (University of Reading, U.K.). Sponsored by COST (European Cooperation in the field of Scientific and Technical Research), the European Science Foundation and the European Office of Aerospace Research and Development.
The work has been supported by EU 5th Framework Programme projects (QLK5-1999-01267, QLK3-2000-01528 and QLK3-2001-01676) and by domestic sources (OTKA, FKFP, OMFB and OM KFHÁT). International collaboration within the EU network COST Action 841 is greatly appreciated.