Classification and phylogeny of hydrogenases.

Hydrogenases (H2ases) catalyze the reversible oxidation of molecular hydrogen and play a central role in microbial energy metabolism. Most of these enzymes are found in Archaea and Bacteria, but a few are present in Eucarya as well. They can be distributed into three classes: the [Fe]-H2ases, the [NiFe]-H2ases, and the metal-free H2ases. The vast majority of known H2ases belong to the first two classes, and over 100 of these enzymes have been characterized genetically and/or biochemically. Compelling evidence from sequences and structures indicates that the [NiFe]- and [Fe]-H2ases are phylogenetically distinct classes of proteins. The catalytic core of the [NiFe]-H2ases is a heterodimeric protein, although additional subunits are present in many of these enzymes. Functional classes of [NiFe]-H2ases have been defined, and they are consistent with categories defined by sequence similarity of the catalytic subunits. The catalytic core of the [Fe]-H2ases is a ca. 350-residue domain that accommodates the active site (H-cluster). A few monomeric [Fe]-H2ases are barely larger than the H-cluster domain. Many others are monomeric as well, but possess additional domains that contain redox centers, mostly iron-sulfur. Some [Fe]-H2ases are oligomeric. The modular structure of H2ases is strikingly illustrated in recently unveiled sequences and structures. It is also remarkable that most of the accessory domains and subunits of H2ases have counterparts in other redox complexes, in particular NADH-ubiquinone oxidoreductase (Complex I) of respiratory chains. Microbial genome sequences are bringing forth a significant body of additional H2ase sequence data and contribute to the understanding of H2ase distribution and evolution. Altogether, the available data suggest that [Fe]-H2ases are restricted to Bacteria and Eucarya, while [NiFe]-H2ases, with one possible exception, seem to be present only in Archaea and Bacteria. H2ase processing and maturation involve the products of several genes which have been identified and are currently being characterized in the case of the [NiFe]-H2ases. In contrast, near to nothing is known regarding the maturation of the [Fe]-H2ases. Inspection of the currently available genome sequences suggests that the [NiFe]-H2ase maturation proteins have no similar counterparts in the genomes of organisms possessing [Fe]-H2ases only. This observation, if confirmed, would be consistent with the phylogenetic distinctiveness of the two classes of H2ases. Sequence alignments of catalytic subunits of H2ases have been implemented to construct phylogenetic trees that were found to be consistent, in the main, with trees derived from other data. On the basis of the comparisons performed and discussed here, proposals are made to simplify and rationalize the nomenclature of H2ase-encoding genes.

Molecular biology studies of the uptake hydrogenase of Rhodobacter capsulatus and Rhodocyclus gelatinosus.

In the photosynthetic bacteria, as in other N2-fixing bacteria, two main enzymes are involved in H2 metabolism: nitrogenase, which catalyses the photoproduction of H2, and a membrane-bound (NiFe) hydrogenase, which functions as an H2-uptake enzyme. The structural genes for Rhodobacter capsulatus and Rhodocyclus gelatinosus uptake hydrogenases were isolated and sequenced. They present the same organization, with the gene encoding the small subunit (hupS) (molecular masses 34.2 and 34.6 kDa, respectively) preceding the gene encoding the large one (hupL) (molecular masses 65.8 and 68.5 kDa, respectively). The two hupSL genes apparently belong to the same operon. The deduced protein sequences of the small and of the large subunits share nearly 80% and maximally 70% identity, respectively, with their counterparts in uptake hydrogenases found in N2-fixing bacteria. However, unlike in Bradyrhizobium japonicum, R. gelatinosus or Azotobacter chroococcum, another open reading frame (ORFX) was found downstream and contiguous to the R. capsulatus hupSL whose transcription seemed to depend on the same hup promoter as hupSL. ORFX contained 786 nucleotides capable of encoding a hydrophobic polypeptide of 262 amino acids (30.2 kDa).

A manganese-containing superoxide dismutase from Paracoccus denitrificans.

A cyanide-insensitive superoxide dismutase (superoxide: superoxide dismutase EC 1.15.1.1) has been isolated from Paracoccus denitrificans, purified to homogeneity and characterized. It is a soluble, manganese-containing protein with an apparent molecular weight of 41 500 +/- 1000. It is composed of two identical subunits (Mr 23 500) not bound by disulfide linkage. It's isoelectric point is 4.5. The amino acid composition shows strong similarities with other dimeric procaryotic and with tetrameric mitochondrial Mn-superoxide dismutases. The fully active enzyme contained from 1.34 to 2 gatom Mn/mol enzyme.

Comparison of the membrane-bound and detergent-solubilised hydrogenase from paracoccus denitrificans. Isolation of the hydrogenase.

The hydrogenase from Paracoccus denitrificans is an integral membrane protein and has been solubilised by Triton X-100. The membrane-bound and detergent-solubilised forms of the enzyme have been compared. Both forms of the enzyme show a pH optimum for reduction of benzyl viologen at pH 8.5--9.0 and are both inhibited by concentrations of NaCl greater than 30 mM. An Arrhenius plot of the activity of hydrogenase in the membrane shows no 'break'. The form of the Arrhenius plot and the activation energy are not significantly changed on solubilisation of the enzyme. The Km and V values for benzyl viologen, methyl viologen and H2 are unaltered when the enzyme is extracted from the membrane. Therefore, solubilisation of hydrogenase from the membrane by Triton X-400 is unlikely to disrupt the native conformation of the enzyme. The detergent-solubilised hydrogenase has subsequently been purified using ammonium sulphate precipitation, sucrose density gradient centrifugation and chromatography on hydroxyapatite. The overall yield of activity is 23%, with a final purification of over 100-fold.

Functional relationship between the ADP/ATP-carrier and the F1-ATPase in mitochondria.

1. The distribution of labeled and unlabeled adenine-nucleotides inside and outside mitochondria was followed after addition of [14C]ADP to rat liver mitochondria. Two types of mitochondria were used: 1, respiring mitochondria which were carrying out oxidative phosphorylation and which had been replenished in ATP by incubation in a medium supplemented with succinate and phosphate; 2, non-respiring mitochondria which had been partially depleted of ATP by incubation in a medium supplemented with rotenone and phosphate. During the first minute following addition of [14C]ADP to the respiring mitochondria, the pre-existing intramitochondrial (internal) [12C]ATP was released into the medium and replaced by newly synthesized [14C]ATP. No [14C]ADP accumulated in the mitochondria. It is suggested that extramitochondrial (external) ADP entering respiring mitochondria in exchange for internal ATP is phosphorylated to ATP before its complete release in the matrix space. In non-respiring mitochondria, the entry of [14C]ADP into the mitochondria was accompanied by the appearance in the external space of [12C]ADP and [12C]ATP, with a marked predominance of [12C]ADP. Thus in non-respiring mitochondria, the residual internal ATP is dephosphorylated to ADP in the inner membrane before being released outside the mitochondria. 2. When mitochondria were incubated with glutamate, ADP and [32P]phosphate, the [32P]ATP which accumulated in the matrix space became rapidly labeled in both the P gamma and P beta groups of the ATP, due to the presence of a transphosphorylation system in the mitochondrial matrix. The [32P]ATP which accumulated outside the mitochondria was also labeled in the P beta group, although less rapidly than the internal ATP. Our data show that a large fraction (75-80%) of the ATP produced by phosphorylation of added ADP within the inner mitochondrial membrane is released into the matrix space before being transported out from the mitochondria; only a small part (20-25%) is released directly outside the mitochondria without penetrating the matrix space. 3. In respiring and phosphorylating mitochondria, the value of the Km of the ADP-carrier for external ADP was 2-4 times lower than its value in non-respiring and non-phosphorylating mitochondria. 4. The above experimental data are discussed with reference to the topological and functional relationships between the ADP-carrier and the oxidative phosphorylation complex in the inner mitochondrial membrane. They strongly suggest that the ADP-carrier comes to the close neighbourhood of the ATP synthetase on the matrix side of the inner membrane.

Binding of radioactively labeled carboxyatractyloside, atractyloside and bongkrekic acid to the ADP translocator of potato mitochondria.

1. The inhibition of the ADP-stimulated respiration of potato mitochondria by carboxyatractyloside is relieved by high concentration of ADP or by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Atractyloside is a much less potent inhibitor than carboxyatractyloside. The inhibition of the ADP-stimulated respiration required about 60-times more atractyloside than carboxyatractyloside. 2. [35S]carboxyatractyloside and [3H]bongkrekic acid bind to potato mitochondria with high affinity (Kd = 10 to 20 nM, n=0.6-0.7 nmol per mg protein). Added ADP competes with carboxyatractyloside for binding; on the contrary ADP increases the amount of bound bongkrekic acid. [3H]atractyloside binds to potato mitochondria with a much lower affinity (Kd=0.45 muM) than carboxyatractyloside or bongkrekic acid. 3. Bound [3H]atractyloside is displaced by ADP, carboxyatractyloside and bongkrekic acid. The displacement of bound [35S]carboxyatractyloside by bongkrekic acid and of bound [3H]bongkrekic acid by carboxyatractyloside is markedly increased by ADP. 4. Bongkrekic acid competes with [35S]carboxyatractyloside for binding. Addition of a small concentration of ADP considerably enhances the inhibitory effect of bongkrekic acid on [35S]carboxyatractyloside binding. 5. The adenine nucleotide content of potato mitochondria is of the order of 1 nmol per mg protein. ADP transport in potato mitochondria is inhibited by atractyloside 30- to 40-times less efficiently than by carboxyatractyloside.

Study of the transverse diffusion of spin-labeled phospholipids in biological membranes. II. Inner mitochondrial membrane of rat liver: use of phosphatidylcholine exchange protein.

Spin-labeled phosphatidylcholine was incorporated into the membrane of isolated "inner membrane+matrix" particles of rat liver mitochondria by incubation with sonicated spin-labeled phosphatidylcholine vesicles at 22 degrees C. When the spin label was on the acyl chain the incorporation of phosphatidylcholine into the membrane was stimulated by the presence of the phosphatidylcholine exchange protein extracted from rat or beef liver. On the other hand no stimulation was observed when the nitroxide was on the polar head-group. When spin-labeled phosphatidycholine was incorporated into the mitochondrial membrane in the absence of phosphatidylcholine exchange protein, ascorbate treatment at 0 degrees C reduced the EPR signal of the spin-labeled membranes by approximately 50%, indicating that fusion incorporates molecules equally on both sides of the membrane. On the other hand when spin-labeled phosphatidylcholine was incorporated in the presence of the exchange protein most of the EPR signal could be destroyed by the ascorbate treatment at 0 degrees C, indicating that the spin-labeled phosphatidylcholine had been selectively incorporated in the outer layer of the membrane. Finally when the label is on the polar head-group the inner content of mitochondria reduces the label facing the matrix, thus creating again an anisotropy of the labeling. The anisotropic distribution of spin-labeled phosphatidylcholine in the mitochondrial membrane was found to be stable at 25 degrees C for more than 2 h. It is therefore concluded that the rate of outside-inside and inside-outside transitions are extremely slow (half-life greater than 24 h).

Isolation and characterization of a protein with cyanide-sensitive superoxide dismutase activity from the prokaryote, Paracoccus denitrificans.

1. A protein with cyanide-sensitive superoxide dismutase activity was isolated from the prokaryote Paracoccus denitrificans. 2. This enzyme, present in low amount in the cell, represented not more than 10% of the total cellular superoxide dismutase activity. It was obtained in a form which was 20-40-times less active than the main superoxide dismutase of P. denitrificans which is a manganese-containing enzyme. 3. It was a soluble monomeric enzyme, highly negatively charged (pI = 4.8), with an apparent molecular weight of 33,000. 4. Cyanide sensitivity was observed by NMR assay, enzyme assay and by staining the protein for superoxide dismutase activity on polyacrylamide electrophoretogram. KCN was shown to be a competitive inhibitor of this dismutase, with an inhibitor constant of 0.15 mM. 5. From the amino acid analysis, S delta Q values lower than 100 were obtained with copper-containing proteins such as the subunit II of cytochrome oxidase from P. denitrificans (69), the azurin from P. denitrificans (77), the bacteriocuprein from Photobacter leiognathi (71); with iron and manganese superoxide dismutases (40-88), and with some eukaryotic copper/zinc dismutases of fish origin (55-82).

Structural study of the response regulator HupR from Rhodobacter capsulatus. Electron microscopy of two-dimensional crystals on a nickel-chelating lipid.

Two-dimensional crystals of the histidine-tagged-HupR protein, a transcriptional regulator from the photosynthetic bacterium Rhodobacter capsulatus, were obtained upon specific interaction with a Ni2+-chelated lipid monolayer. HupR is a response regulator of the NtrC family; it activates the transcription of the structural genes, hupSLC, of the [NiFe]hydrogenase. The lipid (Ni-NTA-DOGA) uses the metal chelator nitrilotriacetic group as the hydrophilic headgroup and contains unsaturated oleyl tails to provide the fluidity necessary for two-dimensional protein crystallization. A projection map of the full-length protein at 18 A resolution was generated by analysing electron microscopy micrographs of negatively stained crystals. The HupR protein appeared to be dimeric and revealed a characteristic "propeller-like" motif. Each monomer forms an L-shaped structure.

Cloning and sequence analyses of the genes coding for the integration host factor (IHF) and HU proteins of Pseudomonas aeruginosa.

Histone-like proteins, such as HU and the integration host factor (IHF), are small, dimeric, DNA-bending proteins which play a role in maintaining constrained DNA structures and hence in regulating gene expression. Two different strategies were used to isolate the genes coding for Pseudomonas aeruginosa (Pa) HU and IHF, two proteins that we have previously isolated from a mucoid strain. By use of a PCR-based technique with oligodeoxyribonucleotides (oligos) designed from the N-terminal amino acid (aa) sequences of HU and the beta-subunit of IHF, and Southern blot analyses, hupB and himD, encoding HU and IHF beta, respectively, have been cloned. The himA gene of Pa, encoding the alpha-subunit of IHF, was isolated using himA of Escherichia coli (Ec) as a probe in Southern blot analyses. The deduced hupB product (90 aa, 9 kDa) is 79% identical to HU beta and 61% to HU alpha of Ec. The predicted products of himA (100 aa, 11.5 kDa) and of himD (94 aa, 10.6 kDa) share 77 and 70% identity with IHF alpha and IHF beta of Ec, respectively. The promoter region of himD contains an IHF consensus sequence, as is the case for Ec himD.

Cloning and sequence of the structural (hupSLC) and accessory (hupDHI) genes for hydrogenase biosynthesis in Thiocapsa roseopersicina.

The first molecular biology study on the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina is reported, namely, the construction of cosmid libraries and isolation of a hydrogenase gene cluster by hybridization with hydrogenase structural genes from the purple non-sulfur bacterium, Rhodobacter capsulatus. The sequenced gene cluster contains six open reading frames, the products of which show significant degrees of identity (from 40 to 78%) with hydrogenase gene products necessary for biosynthesis of the group-I of [NiFe]hydrogenases. The structural hupSLC genes encode the small and large hydrogenase subunits and a hydrophobic protein shown to accept electrons from hydrogenase in R. capsulatus. They are followed downstream by three genes, hupDHI, which are similar to hydrogenase accessory genes found in other bacteria.

Isolation of Rhodobacter capsulatus transketolase: cloning and sequencing of its structural tktA gene.

Rhodobacter capsulatus transketolase (Tkt) protein has been isolated from strain B10 by heparin affinity chromatography. Oligodeoxyribonucleotides (oligo) constructed as based on the amino-acid sequences were used for polymerase chain reaction (PCR) amplification on total genomic DNA. Southern hybridization with the PCR product as a probe allowed the isolation of a 5-kb PstI DNA fragment containing the structural Tkt-encoding gene (tktA) which was cloned and sequenced. The deduced tktA product of 671 aa (72815 Da) shares 59% identity with Rhodobacter sphaeroides Tkt.

Characterisation of the mcpA and mcpB genes capable of encoding methyl-accepting type chemoreceptors in Rhodobacter capsulatus.

Two contiguous mcp genes, mcpA and mcpB, transcribed from the same DNA strand and capable of encoding methyl-accepting chemotaxis proteins (Mcp) have been isolated from Rhodobacter capsulatus (Rc), sequences and overexpressed in Escherichia coli (Ec). The deduced proteins (McpA, 69 171 Da; McpB, 81 629 Da) show a structure similar to that of Ec Mcp. The products of mcpA and mcpB, overproduced in Ec, were recognized by anti-Ec Mcp (Trg) antibodies.

Nitrogen fixation and hydrogen metabolism in photosynthetic bacteria.

The photosynthetic bacteria are found in a wide range of specialized aquatic environments. These bacteria represent important members of the microbial community since they are capable of carrying out two of the most important processes on earth, namely, photosynthesis and nitrogen fixation, at the expense of solar energy. Since the discovery that these bacteria could fix atmospheric nitrogen, there has been an intensification of studies relating to both the biochemistry and physiology of this process. The practical importance of this field is emphasized by a consideration of the tremendous energy input required for the production of artificial nitrogenous fertilizer. The present communication aims to briefly review the current state of knowledge relating to certain aspects of nitrogen fixation by the photosynthetic bacteria. The topics that will be discussed include a general survey of the nitrogenase system in the various photosynthetic bacteria, the regulation of both nitrogenase biosynthesis and activity, recent advances in the genetics of the nitrogen fixing system, and the hydrogen cycle in these bacteria. In addition, a brief discussion of some of some of the possible practical applications provided by the photosynthetic bacteria will be presented.

Cloning of DNA fragments carrying hydrogenase genes of Rhodopseudomonas capsulata.

A cosmid library of Rhodopseudomonas capsulata DNA was constructed in Escherichia coli HB101 using the broad-host-range cosmid vector pLAFR1. More than ninety per cent of the clones in the bank contained cosmids with DNA inserts averaging 20 kilobase pairs in length. Mutants deficient in uptake hydrogenase (Hup-) were obtained from R. capsulata strain B10 by ethylmethylsulfonate (EMS) mutagenesis. The content of hydrogenase protein in Hup- mutant cells was tested by rocket immunoelectrophoresis. Hup- mutants (Rifr) were complemented with the clone bank by conjugation and, from the transconjugants selected by rifampicin and tetracycline resistance, Hup+ transconjugants were screened for the ability to grow photoautotrophically and to reduce methylene blue in a colony assay. The recombinant plasmid pAC57 restored hydrogenase activity in the Hup- mutants RCC8, RCC10, RCC12 and ST410 whereas pAG202 restored that of IR4. The cloned R. capsulata DNA insert of pAC57 gave 5 restriction fragments by cleavage with EcoRI endonuclease. Fragment 1 (7 kb) restored hydrogenase activity in Hup- mutant strains RCC12 and ST410 and fragment 5 (1.3 kb) in strains RCC8 and RCC10. Since the 2 cosmids pAC57 and pAG202 are different cosmids, as indicated by restriction analyses and absence of cross hybridization, it is concluded that at least two hup genes are required for the expression of hydrogenase activity in R. capsulata.

Are monoglyceride-lipase, triglyceride-lipase and phospholipase A of rat liver microsomes distinct protein entities?

1. Different extraction and purification techniques were employed for the separation of MG-lipase, TG-lipase and phospholipase A from rat liver microsomes. 2. Up to 60 per cent of the microsomal content of TG-lipase and phospholipase could be extracted with 1 M KCl or NaCl. MG-lipase was extracted more readily by detergents (e.g. emulphogen). 3. MG-lipase is more resistant to detergent and heat inactivation than TG-lipase and phospholipase A. It is retained, using the technique of affinity chromatography, on a column of CH-Sepharose coupled to monooleoylglycerol. In addition, MG-lipase was separated from TG-lipase by electrofocusing. 4. TG-lipase and phospholipase A were partially separated by gel filtration on Sephadex G-200 in the presence of 1 mM dithiothreitol and by chromatography on CH-Sepharose 4b. 5. On the basis of the present extraction and purification studies, it is concluded that mg-lipase is an enzyme protein distinct from TG-lipase and phospholipase A.

The IHF proteins of Rhodobacter capsulatus and Pseudomonas aeruginosa.

The binding properties of the two IHF consensus sequences present in the promoter region of the hydrogenase structural operon, hupSL, of Rhodobacter capsulatus were studied by gel retardation assays using the heterodimeric IHF-like proteins isolated from R capsulatus, from Pseudomonas aeruginosa and from Escherichia coli. The three IHF proteins bound preferentially to the IHF consensus proximal to hupS. The three-dimensional structure of R capsulatus IHF was modeled using a computer-based amino acid replacement strategy and the known coordinates of crystallized HU protein (HBS) from Bacillus stearothermophilus. Double-stranded DNA and the interaction of IHF and DNA were then modeled using the molecular modeling package Quanta 3.3, and taking into account foot-printing data obtained with IHF-DNA complexes and the fact that the replacement of Arg8 by Cys8 in the alpha subunit, the product of himA, renders R capsulatus IHF ineffective in the activation of hydrogenase synthesis. In this model, IHF is shown to interact with DNA bent by 140 degrees, and Arg8 of HimA capable of interacting with the phosphate-ribose backbone of DNA in the flanking region of the IHF binding site.

Identification of the rpmF-plsX-fabH genes of Rhodobacter capsulatus.

The rpmF-plsX-fabH gene cluster of Rhodobacter capsulatus homologous to that of Escherichia coli was identified. rpmF encodes ribosomal protein L32, plsX plays an undefined role in membrane lipid synthesis, and fabH encodes beta-ketoacyl-acyl carrier protein synthase III. The R. capsulatus plsX gene complemented a defect in an E. coli strain with the plsX50 mutation. Overproduction of the fabH gene product of R. capsulatus in E. coli resulted in dramatically increased beta-ketoacyl-acyl carrier protein synthase III activity. These results indicate that plsX and fabH apparently function the same in R. capsulatus as in E. coli.