Localization of the membrane-bound hydrogenase in Alcaligenes eutrophus by electron microscopic immunocytochemistry

Abstract The membrane-bound hydrogenase was localized in cells of Alcaligenes eutrophus by electron microscopic immunocytochemistry. Post-embedding labeling performed on ultrathin sections revealed that the enzyme was located predominantly (80%) at the cell periphery in autotrophically and heterotrophically grown bacteria harvested from the exponential phase of growth. In the stationary growth phase, however, only 50% of the enzyme was found at the cell periphery; the remaining 50% was distributed over the cytoplasm. The relative amount of electron microscopic label per cell as seen by application of the protein A—gold technique was higher in cells grown autotrophically as compared to cells grown heterotrophically on fructose. Derepression of the enzyme was followed electron microscopically in a substrate-shift experiment (growth on fructose, followed by a shift to glycerol). Major amounts of the enzyme appeared to undergo a reattachment to the cytoplasmic membrane under these conditions, starting with a reduced location of the enzyme in the cytoplasm and an accumulation in cell areas close to the cytoplasmic membrane. These findings indicate that the 'membrane-bound' hydrogenase (i.e., that material enriched as membrane-bound enzyme according to the appropriate activity test) is not, in fact, membrane bound or membrane integrated but membrane associated. It may or may not interact with the cytoplasmic face of the cytoplasmic membrane, depending on the growth phase and conditions.     

Maturation of membrane-bound hydrogenase of Alcaligenes eutrophus H16.

The formation of the catalytically active membrane-bound hydrogenase (MBH) of Alcaligenes eutrophus H16 requires the genes for the small and large subunits of the enzyme (hoxK and hoxG, respectively) and an accompanying set of accessory genes (C. Kortl ke, K. Horstmann, E. Schwartz, M. Rohde, R. Binsack, and B. Friedrich, J. Bacteriol. 174:6277-6289, 1992). Other genes located in the adjacent pleiotropic region are also required. In the absence of these genes, MBH is synthesized but is catalytically inactive. Immunological analyses revealed that cells containing active MBH produced the small and large subunits of the enzyme in two distinct conformations each; only one of each, presumably the immature form, occurred in cells devoid of MBH activity. The results suggest that the conversion of the two subunits into the catalytically active membrane-associated heterodimer depends on specific maturation processes mediated by hox genes.     

The membrane-bound hydrogenase of Alcaligenes eutrophus. I. Solubilization, purification, and biochemical properties.

The membrane-bound hydrogenase of Alcaligenes eutrophus was solubilized from washed membranes of autotrophically grown cells. The enzyme consists of two types of subunits and is an iron-sulfur protein. A flavin compound was not detected. The enzyme reacts only with few artificial electron acceptors.     

Antigenic determinants of the membrane-bound hydrogenase in Alcaligenes eutrophus are exposed toward the periplasm.

Electron microscopic immunogold labeling experiments were performed with ultrathin sections of plasmolyzed cells of Alcaligenes eutrophus and "whole-mount" samples of spheroplasts and protoplasts. They demonstrated that antigenic determinants of the membrane-bound hydrogenase are exposed, at the outside of the cytoplasmic membrane, to the periplasm.     

The iron-sulphur centres of soluble hydrogenase from Alcaligenes eutrophus.

The soluble hydrogenase (hydrogen:NAD+ oxidoreductase (EC 1.12.1.2) from Alcaligenes eutrophus has been purified to homogeneity by an improved procedure, which includes preparative electrophoresis as final step. The specific activity of 57 mumol H2 oxidized/min per mg protein was achieved and the yield of pure enzyme from 200 g cells (wet weight) was about 16 mg/purification. After removal of non-functional iron, analysis of iron and acid-labile sulphur yielded average values of 11.5 and 12.9 atoms/molecule of enzyme, respectively. p-Chloromercuribenzoate was a strong inhibitor of hydrogenase and apparently competed with NAD not with H2. Chelating agents, CO and O2 failed to inhibit enzyme activity. The oxidized hydrogenase showed an EPR spectrum with a small signal at g = 2.02. On reduction the appearance of a high temperature (50--77 K) signal at g = 2.04, 1.95 and a more complex low temperature (less than 30 K) spectrum at g = 2.04, 2.0, 1.95, 1.93, 1.86 was observed. The pronounced temperature dependence and characteristic lineshape of the signals obtained with hydrogenase in 80--85% dimethylsulphoxide demonstrated that iron-sulphur centres of both the [2Fe-2S] and [4Fe-4S] types are present in the enzyme. Quantitation of the EPR signals indicated the existence of two identical centres each of the [4Fe-4S] and of the [2Fe-2S] type. The midpoint redox potentials of the [4Fe-4S] and the [2Fe-2S] centres were determined to be -445 mV and -325 mV, respectively. Spin coupling between two centres, indicated by the split feature of the low temperature spectrum of the native hydrogenase around g = 1.95, 1.93, has been established by power saturation studies. On reduction of the [Fe-4S] centres, the electron spin relaxation rate of the [2Fe-2S] centres was considerably increased. Treatment of hydrogenase with CO caused no change in EPR spectra.     

The plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus

Abstract Alcaligenes eutrophus strain H16 harbors a 450 kilobase pairs (kb) conjugative plasmid which codes for the ability of the organism to grow lithoautotrophically on hydrogen and carbon dioxide (reviewed in [1]). The genes for hydrogen oxidation, designated hox , are clustered on plasmid pHG1 in a DNA region of approximately 100-kb in size ([2], Fig. 1). The hox genes and their organization have been analyzed by isolation of Hox-deficient mutants, by complementation analysis, by cloning of hox genes, identification of hox -encoded polypeptides and, most recently, by DNA sequencing. The hox cluster is flunked by the two structural gene regions, hoxS and hoxP ; it contains a regulatory locus, hoxC , and additional genes like hoxN and hoxM whose products play a role in the formation of catalytically active hydrogenase proteins. Of four indigenous 1.3-kb insertion elements, two copies of IS491 map in the hox gene cluster. These elements may be involved in rearrangements and deletions which occur particularly frequently in this region of the megaplasmid (Schwartz, Kortlüke and Friedrich, unpublished).     

Molecular and immunological comparison of membrane-bound, H2-oxidizing hydrogenases of Bradyrhizobium japonicum, Alcaligenes eutrophus, Alcaligenes latus, and Azotobacter vinelandii.

The membrane-bound hydrogenases of Bradyrhizobium japonicum, Alcaligenes eutrophus, Alcaligenes latus, and Azotobacter vinelandii were purified extensively and compared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of each hydrogenase revealed two prominent protein bands, one near 60 kilodaltons and the other near 30 kilodaltons. The migration distances during nondenaturing polyacrylamide gel electrophoresis were similar for all except A. vinelandii hydrogenase, which migrated further than the other three. The amino acid composition of each hydrogenase was determined, revealing substantial similarity among these enzymes. This was confirmed by calculation of S delta Q values, which ranged from 8.0 to 26.7 S delta Q units. S delta Q is defined as sigma j(Xi,j-Xk,j)2, where i and k identify the proteins compared and Xj is the content (residues per 100) of a given amino acid of type j. The hydrogenases of this study were also compared with an enzyme-linked immunosorbent assay. Antibody raised against B. japonicum hydrogenase cross-reacted with all four hydrogenases, but to various degrees and in the order B. japonicum greater than A. latus greater than A. eutrophus greater than A. vinelandii. Antibody raised against A. eutrophus hydrogenase also cross-reacted with all four hydrogenases, following the pattern of cross-reaction A. eutrophus greater than A. latus = B. japonicum greater than A. vinelandii. Antibody raised against B. japonicum hydrogenase inhibited B. japonicum hydrogenase activity to a greater extent than the A. eutrophus and A. latus activities; no inhibition of A. vinelandii hydrogenase activity was detected. The results of these experiments indicated remarkable homology of the hydrogenases from these four microorganisms.     

Structural organization of the membrane-bound hydrogenase isolated from Alcaligenes eutrophus as revealed by electron microscopy

Electron microscopy of negatively stained samples of the membrane-bound hydrogenase isolated from Alcaligenes eutrophus was used to obtain enzyme images with an estimated resolution of 2.5 nm. The two subunits with shapes similar to the letter 'U' making up the enzyme could be seen to be joined in two planes orthogonal to each other, making contact with their concave sides. In face-on view, the particle exhibited bilateral symmetry.     

Structural organization of the membrane-bound hydrogenase isolated from Alcaligenes eutrophus as revealed by electron microscopy

Abstract Electron microscopy of negatively stained samples of the membrane-bound hydrogenase isolated from Alcaligenes eutrophus was used to obtain enzyme images with an estimated resolution of 2.5 nm. The two subunits with shapes similar to the letter 'U' making up the enzyme could be seen to be joined in two planes orthogonal to each other, making contact with their concave sides. In face-on view, the particle exhibited bilateral symmetry.     

Comparison of the membrane-bound hydrogenases from Alcaligenes eutrophus H16 and Alcaligenes eutrophus type strain

Whereas the membrane-bound hydrogenase from Alcaligenes eutrophus H16 is an integral membrane protein and can only be solubilized by detergent treatment, the membrane-bound hydrogenase of Alcaligenes eutrophus type strain was found to be present in a soluble form after cell disruption. For the enzyme of A. eutrophus H16 a new, highly effective purification procedure was developed including phase separation with Triton X-114 and triazine dye chromatography on Procion Blue H-ERD-Sepharose. The purification led to an homogeneous hydrogenase preparation with a specific activity of 269 U/mg protein (methylene blue reduction) and a yield of 45%. During purification and storage the enzyme was optimally stabilized by the presence of 0.2 mM MnCl2. The hydrogenase of A. eutrophus type strain was purified from the soluble extract by a similar procedure, however, with less specific activity and activity yield. Comparison of the two purified enzymes revealed no significant differences: They have the same molecular weight, both consist of two different subunits (Mr = 62,000, 31,000) and both have an isoelectric point near pH 7.0. They have the same electron acceptor specificity reacting with similar high rates and similar Km values. The acceptors reduced include viologen dyes, flavins, quinones, cytochrome c, methylene blue, 2,6-dichlorophenolindophenol, phenazine methosulfate and ferricyanide. Ubiquinones and NAD were not reduced. The two hydrogenases were shown to be immunologically identical and both have identical electrophoretic mobility. For the membrane-bound hydrogenase of A. eutrophus H16 it was demonstrated that this type of hydrogenase in its solubilized, purified state is able to catalyze also the reverse reaction, the H2 evolution from reduced methyl viologen.     

Nickel in the catalytically active hydrogenase of Alcaligenes eutrophus.

Nickel is a constituent of soluble and particulate hydrogenase of Alcaligenes eutrophus. Incorporation of 63Ni2+ revealed that almost the total nickel taken up by the cells was bound to the protein. Chromatography of a crude extract on diethylaminoethyl cellulose demonstrated an association of 63Ni2+ with soluble and particulate hydrogenase, supported by further analysis like polyacrylamide gel electrophoresis. Unspecific binding of 63Ni2+ to the protein was excluded by comparison with a mutant extract free of hydrogenase protein. X-ray fluorescence analysis of the homogeneous soluble hydrogenase indicated the presence of 2 mol of nickel per mol of enzyme, whereas the amount of nickel determined by incorporation of 63Ni2+ was calculated to be approximately 1 mol/mol of enzyme. Cells grown under nickel limitation contained catalytically inactive, but serologically active, soluble and particulate hydrogenase. The immunochemical reactions were only partially identical with the enzyme from nickel-cultivated cells indicating a structural modification of the proteins in the absence of nickel. It is concluded that nickel is essential for the catalytic activity of hydrogenase and not involved as a regulatory component in the synthesis of this enzyme.     

Nickel requirement for active hydrogenase formation in Alcaligenes eutrophus.

The nickel-dependent chemolithoautotrophic growth of Alcaligenes eutrophus is apparently due to a requirement of nickel for active hydrogenase formation. Cells grown heterotrophically with fructose and glycerol revealed a specific activity of soluble and membrane-bound hydrogenase which was severalfold higher than the normal autotrophic level. The omission of nickel from the medium did not affect heterotrophic growth, but the soluble hydrogenase activity was reduced significantly. In the presence of ethylenediaminetetraacetic acid (EDTA), almost no hydrogenase activity was detected. The addition of nickel allowed active hydrogenase formation even when EDTA was present. When chloramphenicol was added simultaneously with nickel to an EDTA-containing medium, almost no hydrogenase activity was found. This indicates that nickel ions are involved in a process which requires protein synthesis and not the direct reactivation of a preformed inactive protein. The formation of the membrane-bound hydrogenase also appeared to be nickel dependent. Autotrophic CO2 assimilation did not specifically require nickel ions, since formate was utilized in the presence of EDTA and the activity of ribulosebisphosphate carboxylase was not affected under these conditions.     

In vivo inactivation of soluble hydrogenase of Alcaligenes eutrophus

The soluble, NAD⁺-reducing hydrogenase in intact cells of Alcaligenes eutrophus was inactivated by oxygen when electron donors such as hydrogen or pyruvate were available. The sole presence of either oxygen or oxidizable substrates did not lead to inactivation of the enzyme. Inactivation occurred similarly under autotrophic growth conditions with hydrogen, oxygen and carbon dioxide. The inactivation followed first order reaction kinetics, and the half-life of the enzyme in cells exposed to a gas atmosphere of hydrogen and oxygen (8:2, v/v) at 30° C was 1.5 h. The process of inactivation did not require ATP-synthesis. There was no experimental evidence that the inactivation is a reversible process catalyzed by a regulatory protein. The possibility is discussed that the inactivation is due to superoxide radical anions (O ₂ ^- ) produced by the hydrogenase itself.     

Nickel and iron incorporation into soluble hydrogenase of Alcaligenes eutrophus

Archives of Microbiology - Qualitative and quantitative determination of proteins of the soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H16 was done by...     

Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16.

The soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H 16 was purified 68-fold with a yield of 20% and a final specific activity (NAD reduction) of about 54 mumol H2 oxidized/min per mg protein. The enzyme was shown to be homogenous by polyacrylamide gel electrophoresis. Its molecular weight and isoelectric point were determined to be 205 000 and 4.85 respectively. The oxidized hydrogenase, as purified under aerobic conditions, was of high stability but not reactive. Reductive activation of the enzyme by H2, in the presence of catalytic amounts of NADH, or by reducing agents caused the hydrogenase to become unstable. The purified enzyme, in its active state, was able to reduce NAD, FMN, FAD, menaquinone, ubiquinone, cytochrome c, methylene blue, methyl viologen, benzyl viologen, phenazine methosulfate, janus green, 2,6-dichlorophenoloindophenol, ferricyanide and even oxygen. In addition to hydrogenase activitiy, the enzyme exhibited also diaphorase and NAD(P)H oxidase activity. The reversibility of hydrogenase function (i.e. H2 evolution from NADH, methyl viologen and benzyl viologen) was demonstrated. With respect to H2 as substrate, hydrogenase showed negative cooperativity; the Hill coefficient was n = 0.4. The apparent Km value for H2 was found to be 0.037 mM. The absorption spectrum of hydrogenase was typical for non-heme iron proteins, showing maxima (shoulders) at 380 and 420 nm. A flavin component could be extracted from native hydrogenase characterized by its absorption bands at 375 and 447 nm and a strong fluorescense at 526 nm.