Incorporation of a bacterial membrane-bound hydrogenase into proteoliposomes.

Membrane-bound nickel-iron hydrogenases from diverse genera of bacteria have been previously characterized and they are closely related. We report the reconstitution of purified Bradyrhizobium japonicum hydrogenase into proteoliposomes by a detergent dialysis method followed by two or three cycles of freeze-thaw. Sedimentation experiments revealed that more than 60% of the H2-uptake activity was particulate when reconstituted into Escherichia coli phospholipids. Sucrose-gradient centrifugation separated hydrogenase activity into two peaks, the less dense of which was phospholipid-associated and turbid, thereby showing successful incorporation. Purified enzyme did not bind to performed phospholipid vesicles, and 1.0 M NaCl failed to remove incorporated hydrogenase. The optimal micellar detergent:phospholipid ratio (rho) value for hydrogenase incorporation was 2.0. Proteoliposomes containing acidic phospholipids were the most effective for incorporation as well as for activity. The artificial electron acceptor specificity was similar for proteoliposomes and for H2-oxidizing membranes from B. japonicum. Proteoliposomes formed under optimal conditions had a broad size distribution centered around 400 nm diameter. Hydrogenase activity in proteoliposomes was partially protected from inactivation by the protein modification reagent diazobenzene sulfonate (DABS) (inactivation t1/2 = 30 min), whereas DABS rapidly inactivated the purified enzyme (t1/2 = 4 min). The latter result indicates protection of a catalytically important site by the phospholipid bilayer. This experimental system should be useful in addressing questions regarding the in vivo situation of bacterial membrane-bound hydrogenases.     

Transport of Ca2+ and Na+ across the chromaffin-granule membrane.

The soluble hydrogenase (hydrogen-NAD+ oxidoreductase, EC 1.12.1.2) of Alcaligenes eutrophus H16 was shown to be stabilized by oxidation with oxygen and ferricyanide as long as electron donors and reducing compounds were absent. The simultaneous presence of H2, NADH and O2 in the enzyme solution, however, caused an irreversible inactivation of hydrogenase that was dependent on the O2 concentration. The half-life periods of 4 degrees C under partial pressures of 0.1, 5, 20 and 50% O2 were 11, 5, 2.5 and 1.5 h respectively. Evidence has been obtained that hydrogenase produces superoxide free radical anions (O2-.), which were detected by their ability to oxidize hydroxylamine to nitrite. The correlation between O2 concentration, nitrite formation and inactivation rates and the stabilization of hydrogenase by addition of superoxide dismutase indicated that superoxide radicals are responsible for enzyme inactivation. During short-term activity measurements (NAD+ reduction, H2 evolution from NADH), hydrogenase activity was inhibited by O2 only very slightly. In the presence of 0.7 mM-O2 an inhibition of about 20% was observed.     

Purification and characterization of the hydrogen uptake hydrogenase from the hyperthermophilic archaebacterium Pyrodictium brockii.

Pyrodictium brockii is a hyperthermophilic archaebacterium with an optimal growth temperature of 105 degrees C. P. brockii is also a chemolithotroph, requiring H2 and CO2 for growth. We have purified the hydrogen uptake hydrogenase from membranes of P. brockii by reactive red affinity chromatography and sucrose gradient centrifugation. The molecular mass of the holoenzyme was 118,000 +/- 19,000 Da in sucrose gradients. The holoenzyme consisted of two subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large subunit had a molecular mass of 66,000 Da, and the small subunit had a molecular mass of 45,000 Da. Colorometric analysis of Fe and S content in reactive red-purified hydrogenase revealed 8.7 +/- 0.6 mol of Fe and 6.2 +/- 1.2 mol of S per mol of hydrogenase. Growth of cells in 63NiCl2 resulted in label incorporation into reactive red-purified hydrogenase. Growth of cells in 63NiCl2 resulted in label incorporation into reactive red-purified hydrogenase. Temperature stability studies indicated that the membrane-bound form of the enzyme was more stable than the solubilized purified form over a period of minutes with respect to temperature. However, the membranes were not able to protect the enzyme from thermal inactivation over a period of hours. The artificial electron acceptor specificity of the pure enzyme was similar to that of the membrane-bound form, but the purified enzyme was able to evolve H2 in the presence of reduced methyl viologen. The Km of membrane-bound hydrogenase for H2 was approximately 19 microM with methylene blue as the electron acceptor, whereas the purified enzyme had a higher Km value.     

The membrane-bound hydrogenase from Paracoccus denitrificans. Purification and molecular characterization

The membrane-bound hydrogenase from Paracoccus denitrificans was purified 68-fold with a yield of 14.6%. The final preparation had a specific activity of 161.9 mumol H2 min-1 (mg protein)-1 (methylene blue reduction). Purification involved solubilization by Triton X-114, phase separation, chromatography on DEAE-Sephacel, ammonium-sulfate precipitation and chromatography on Procion-red HE-3B-Sepharose. Gel electrophoresis under denaturing conditions revealed two non-identical subunits with molecular masses of 64 kDa and 34 kDa. The molecular mass of the native enzyme was 100 kDa, as estimated by FPLC gel filtration in the presence of Chaps, a zwitterionic detergent. The isoelectric point of the Paracoccus hydrogenase was 4.3. Metal analysis of the purified enzyme indicated a content of 0.6 nickel and 7.3 iron atoms/molecule. ESR spectra of the reduced enzyme exhibited a close similarity to the membrane-bound hydrogenase from Alcaligenes eutrophus H16 with g values of 1.86, 1.92 and 1.98. The half-life for inactivation under air at 20 degrees C was 8 h. The Paracoccus hydrogenase reduced several electron acceptors, namely methylene blue, benzyl viologen, methyl viologen, menadione, cytochrome c, FMN, 2,6-dichloroindophenol, ferricyanide and phenazine methosulfate. The highest activity was measured with methylene blue (V = 161.9 U/mg; Km = 0.04 mM), whereas benzyl and methyl viologen were reduced at distinctly lower rates (16.5 U/mg and 12.1 U/mg, respectively). The native hydrogenase from P. denitrificans cross-reacted with purified antibodies raised against the membrane-bound hydrogenase from A. eutrophus H16. The corresponding subunits from both enzymes also showed immunological relationship. All reactions were of partial identity.     

Chemical Modification of Catalytically Essential Functional Groups of NAD-Dependent Hydrogenase from Ralstonia eutropha H16

Amino acid residues His and Cys of the NAD-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha H16 were chemically modified with specific reagents. The modification of His residues of the nonactivated hydrogenase resulted in decrease in both hydrogenase and diaphorase activities of the enzyme. Activation of NADH hydrogenase under anaerobic conditions additionally modified a His residue (or residues) significant only for the hydrogenase activity. The rate of decrease in the diaphorase activity was unchanged. The modification of thiol groups of the nonactivated enzyme did not affect the hydrogenase activity. The effect of thiol-modifying agents on the activated hydrogenase was accompanied by inactivation of both diaphorase and hydrogenase activities. The modification degree and changes in the corresponding catalytic activities depended on conditions of the enzyme activation. Data on the modification of cysteine and histidine residues of the hydrogenase suggested that the enzyme activation should be associated with significant conformational changes in the protein globule.     

Characterization of the oxygen tolerance of a hydrogenase linked to a carbon monoxide oxidation pathway in Rubrivivax gelatinosus

A hydrogenase linked to the carbon monoxide oxidation pathway in Rubrivivax gelatinosus displays tolerance to O2. When either whole-cell or membrane-free partially purified hydrogenase was stirred in full air (21% O2, 79% N2), its H2 evolution activity exhibited a half-life of 20 or 6 h, respectively, as determined by an anaerobic assay using reduced methyl viologen. When the partially purified hydrogenase was stirred in an atmosphere containing either 3.3 or 13% O2 for 15 min and evaluated by a hydrogen-deuterium (H-D) exchange assay, nearly 80 or 60% of its isotopic exchange rate was retained, respectively. When this enzyme suspension was subsequently returned to an anaerobic atmosphere, more than 90% of the H-D exchange activity was recovered, reflecting the reversibility of this hydrogenase toward O2 inactivation. Like most hydrogenases, the CO-linked hydrogenase was extremely sensitive to CO, with 50% inhibition occurring at 3.9 microM dissolved CO. Hydrogen production from the CO-linked hydrogenase was detected when ferredoxins of a prokaryotic source were the immediate electron mediator, provided they were photoreduced by spinach thylakoid membranes containing active water-splitting activity. Based on its appreciable tolerance to O2, potential applications of this hydrogenase are discussed.     

Purification of the membrane-bound hydrogenase of Escherichia coli.

The membrane-bound hydrogenase (EC class 1.12) of aerobically grown Escherichia coli cells was solubilized by treatment with deoxycholate and pancreatin. The enzyme was further purified to electrophoretic homogeneity by chromoatographic methods, including hydrophobic-interaction chromatography, with a yield of 10% as judged by activity and an overall purification of 2140-fold. The hydrogenase was a dimer of identical subunits with a mol.wt. of 113,000 and contained 12 iron and 12 acid-labile sulphur atoms per molecule. The epsilon 400 was 49,000M-1 . cm-1. The hydrogenase catalysed both H2 evolution and H2 uptake with a variety of artificial electron carriers, but would not interact with flavodoxin, ferredoxin or nicotinamide and flavin nucleotides. We were unable to identify any physiological electron carrier for the hydrogenase. With Methyl Viologen as the electron carrier, the pH optimum for H2 evolution and H2 uptake was 6.5 and 8.5 respectively. The enzyme was stable for long periods at neutral pH, low temperatures and under anaerobic conditions. The half-life of the hydrogenase under air at room temperature was about 12 h, but it could be stabilized by Methyl Viologen and Benzyl Viologen, both of which are electron carriers for the enzyme, and by bovine serum albumin. The hydrogenase was strongly inhibited by carbon monoxide (Ki = 1870Pa), heavy-metal salts and high concentrations of buffers, but was resistant to inhibition by thiol-blocking and metal-complexing reagents. These aerobically grown E. coli cells lacked formate hydrogenlyase activity and cytochrome c552.     

Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12.

Two membrane-bound hydrogenase isoenzymes present in Escherichia coli during anaerobic growth have been resolved. The isoenzymes are immunologically and electrophoretically distinct. The physically more abundant isoenzyme (hydrogenase 1) contains a subunit of Mr 64,000 and is not released from the membrane by exposure to either trypsin or pancreatin. The second isoenzyme (hydrogenase 2) apparently contributes the greater part of the membrane-bound hydrogen:benzyl viologen oxidoreductase activity and exists in two electrophoretic forms revealed by nondenaturing polyacrylamide gel analysis. This isoenzyme is irreversibly inactivated at alkaline pH and gives rise to an active, soluble derivative when the membrane-bound enzyme is exposed to either trypsin or pancreatin. Both hydrogenase isoenzymes contain nickel.     

Topological analysis of the pyridine nucleotide transhydrogenase of Escherichia coli using proteolytic enzymes

The pyridine nucleotide transhydrogenase of Escherichia coli has an alpha 2 beta 2 structure (alpha: Mr, 54,000; beta: Mr, 48,700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the alpha subunit and the N-terminal 20-25 kDa region of the beta subunit are composed of transmembranous alpha-helices. The topology of these subunits in the membrane was investigated using proteolytic enzymes. Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the alpha subunit. The beta subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both alpha and beta subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The beta subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present. NAD(H) did not affect digestion of the beta subunit. Digestion of the beta subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent. It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase. The location of the trypsin cleavage sites in the sequences of the alpha and beta subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the alpha subunit and the C-terminal 30 kDa region of the beta subunit are exposed on the cytoplasmic side of the inner membrane of E. coli. Binding sites for pyridine nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.     

Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii

Uptake hydrogenase (EC 1.12) from Azotobacter vinelandii has been purified 250-fold from membrane preparations. Purification involved selective solubilization of the enzyme from the membranes, followed by successive chromatography on DEAE-cellulose, Sephadex G-100, and hydroxylapatite. Freshly isolated hydrogenase showed a specific activity of 110 mumol of H2 uptake (min X mg of protein)-1. The purified hydrogenase still contained two minor contaminants that ran near the front on sodium dodecyl sulfate-polyacrylamide gels. The enzyme appears to be a monomer of molecular weight near 60,000 +/- 3,000. The pI of the protein is 5.8 +/- 0.2. With methylene blue or ferricyanide as the electron acceptor (dyes such as methyl or benzyl viologen with negative midpoint potentials did not function), the enzyme had pH optima at pH 9.0 or 6.0, respectively, It has a temperature optimum at 65 to 70 degrees C, and the measured half-life for irreversible inactivation at 22 degrees C by 20% O2 was 20 min. The enzyme oxidizes H2 in the presence of an electron acceptor and also catalyzes the evolution of H2 from reduced methyl viologen; at the optimal pH of 3.5, 3.4 mumol of H2 was evolved (min X mg of protein)-1. The uptake hydrogenase catalyzes a slow deuterium-water exchange in the absence of an electron acceptor, and the highest rate was observed at pH 6.0. The Km values varied widely for different electron acceptors, whereas the Km for H2 remained virtually constant near 1 to 2 microM, independent of the electron acceptors.     

Purification and properties of the membrane-bound by hydrogenase from Desulfovibrio desulfuricans.

The membrane-bound hydrogenase from the anaerobic sulphate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) has been purified to homogeneity, with an overall 80-fold purification and a specific activity of 70 mumol of H2 evolved/min per mg of protein. The hydrogenase had a relative molecular mass of 58 000 as determined by gel filtration and was estimated to contain six iron atoms and six acid-labile sulphur groups per molecule. The absorption spectrum of the enzyme was characteristic of an iron-sulphur protein. The E400 and E280 were 28 500 and 109 000 M-1.cm-1 respectively. The e.s.r. of the oxidized protein indicated the presence of [4Fe-4S]3+ or [3Fe-3S]3+, and another paramagnetic centre, probably Ni(III). The hydrogenase was inhibited by heavy-metal salts, carbon monoxide and high ionic strength. However, it was resistant to inhibition by thiol-blocking and metal-complexing reagents. N-Bromosuccinimide totally inhibited the enzyme activity at low concentrations. The enzyme was stable to O2 over long periods and to high temperatures. It catalyses both H2-evolution and H2-uptake with a variety of artificial electron carriers. D. desulfuricans cytochrome C3, its natural electron carrier, had a high affinity for the enzyme (Km = 2 microns). Rate enhancement was observed when cytochrome C3 was added to Methyl Viologen in the H2-evolution assay. The pH optimum for H2-evolution was 6.5.     

Immunological comparison of subunits isolated from various hydrogenases of aerobic hydrogen bacteria

Polyclonal, monospecific antibodies were produced against the two subunits (Mr 62,000, and Mr 31,000), isolated from the membrane-bound hydrogenase of Alcaligenes eutrophus H16. The antibodies (IgG fractions) were purified from crude sera by Protein A-Sepharose CL-4B chromatography. By double immunodiffusion assays and tandem-crossed immunoelectrophoresis the large and the small subunit were demonstrated not to be immunologically related. Immunological comparison of these subunits with the four non-identical subunits (Mr 63,000, 56,000, 30,000 and 26,000) of the NAD-linked, soluble hydrogenase from A. eutrophus H16 showed that the subunits of the membrane-bound hydrogenase did not cross-react with any of the antibodies raised against the four subunits of the NAD-linked enzyme and that, vice versa, none of these four subunits cross-reacted with antibodies raised against the two subunits of the membrane-bound hydrogenase. This means that A. eutrophus H16 contains altogether six non-identical immunologically unrelated hydrogenase polypeptides. The membrane-bound hydrogenases were isolated and purified from various aerobic H2-oxidizing bacteria: A. eutrophus H16, A. eutrophus type strain, A. eutrophus CH34, A. eutrophus Z1, A. hydrogenophilus, Paracoccus denitrificans and strain Cd2/01. All these proteins resembled each other and each consisted of two non-identical polypeptides. A complete separation of these subunits was achieved at high-yield by preparative FPLC gel filtration on three Superose 12 columns connected in series, using SDS and DTT-containing sodium phosphate buffer (pH 7.0). The small subunits of these enzymes turned out to be immunologically closely related to each other; they were either identical or almost identical. The large subunits were also related, but less pronounced. Only the large subunits from Z1 and type strain reacted fully identical with the H16 subunit. Of the two isolated, homogeneous subunits of the membrane-bound hydrogenase from A. eutrophus H16, the amino acid compositions and the NH2-terminal sequences have been determined. The results confirmed the diversity of the large and the small subunit. Furthermore, for comparison also the NH2-terminal sequences of the two subunits from the hydrogenase of A. eutrophus CH34 have been analysed.     

Limited proteolysis by trypsin influences activity of maize phosphoenolpyruvate carboxylase

Maize phosphoenolpyruvate carboxylase (PEPC) was rapidly and completely inactivated by very low concentrations of trypsin at 37 degrees C. PEP+Mg2+ and several other effectors of PEP carboxylase offered substantial protection against trypsin inactivation. Inactivation resulted from a fairly specific cleavage of 20 kDa peptide from the enzyme subunit. Limited proteolysis under catalytic condition (in presence of PEP, Mg2+ and HCO3) although yielded a truncated subunit of 90 kDa, did not affect the catalytic function appreciably but desensitized the enzyme to the effectors like glucose-6-phosphate glycine and malate. However, under non-catalytic condition, only malate sensitivity was appreciably affected. Significant protection of the enzyme activity against trypsin during catalytic phase could be either due to a conformational change induced on substrate binding. Several lines of evidence indicate that the inactivation caused by a cleavage at a highly conserved C-terminal end of the subunit.     

Limited proteolysis of maize NADP-malic enzyme

The incubation of maize malic enzyme at 37 degrees C with trypsin at a ratio of 150:1 of malic enzyme to trypsin caused rapid and complete inactivation of enzyme activity. The inactivation was caused by fairly specific cleavage of the enzyme monomer (62 kDa) into 40 kDa and 20 kDa fragments. The intensity of 40 kDa band increased with the time of treatment of enzyme with trypsin from 2 to 30 min. Substrates, especially NADP (25 microM) provided almost total protection against trypsin inactivation of the enzyme activity. The studies carried out with various other endoproteases indicated that endoprotease Lys-C was most effective in inactivating malic enzyme activity. The kinetic properties of the truncated enzyme have been studied. The Km value for malate in case of native and modified enzyme was found to be identical. Km NADP for the modified enzyme was slightly higher indicating that after proteolysis the enzyme affinity for NADP had decreased. Limited proteolysis with trypsin did not show any appreciable change in fluorescence properties of the modified enzyme. Binding of NADPH to the enzyme was not affected after modification.     

HoxE--a subunit specific for the pentameric bidirectional hydrogenase complex (HoxEFUYH) of cyanobacteria

NAD(P)(+)-reducing hydrogenases have been described to be composed of a diaphorase (HoxFU) and a hydrogenase (HoxYH) moiety. This study presents for the first time experimental evidence that in cyanobacteria, a fifth subunit, HoxE, is part of this bidirectional hydrogenase. HoxE exhibits sequence identities to NuoE of respiratory complex I of Escherichia coli. The subunit composition of the cyanobacterial bidirectional hydrogenase has been investigated. The oxygen labile enzyme complex was purified to close homogeneity under anaerobic conditions from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 6301. The 647-fold and 1290-fold enriched purified enzyme has a specific activity of 46 micromol H(2) evolved (min mg protein)(-1) and 15 micromol H(2) evolved (min mg protein)(-1), respectively. H(2)-evolution of the purified enzyme of S. sp. PCC 6803 is highest at 60 degrees C and pH 6.3. Immunoblot experiments, using a polyclonal anti-HoxE antibody, demonstrate that HoxE co-purifies with the hydrogenase activity in S. sp. PCC 6301. SDS-PAGE gels of the purified enzymes revealed six proteins, which were partially sequenced and identified, besides one nonhydrogenase component, as HoxF, HoxU, HoxY, HoxH and, remarkably, HoxE. The molecular weight of the native protein (375 kDa) indicates a dimeric assembly of the enzyme complex, Hox(EFUYH)(2).     

Controlled tryptic digestion of prostaglandin H synthase. Characterization of protein fragments and enhanced rate of proteolysis of oxidatively inactivated enzyme

Treatment of prostaglandin H (PGH) synthase (70 kDa) with trypsin generates fragments of 33 and 38 kDa. Each of the fragments was purified by reverse-phase high performance liquid chromatography (HPLC) using acetonitrile/water/trifluoroacetic acid gradients. Amino acid sequence analysis indicates that the 33-kDa protein contains the NH2 terminus of PGH synthase. Neither the 33- nor 38-kDa fragment isolated by HPLC exhibits any PGH synthase activity; however, cleavage of intact enzyme to 33- and 38-kDa fragments to the extent of 90% only reduces cyclooxygenase activity by 40%. This implies that the cleaved proteins or a complex formed between them retains the conformation necessary for enzyme activity. Extensive attempts to resolve active fragments from each other or from intact enzyme were unsuccessful; intact enzyme and digestion fragments cochromatograph under all conditions employed. Treatment of PGH synthase with [3H]acetylsalicylic acid followed by trypsin digestion introduces [3H]acetyl moieties into the intact protein and the 38-kDa fragment (0.8-0.9 acetyl group/subunit). Nearly complete conversion of PGH synthase to 33- and 38-kDa fragments by exposure to high concentrations of trypsin prior to [3H]acetylsalicylic acid treatment results in labeling of the 38-kDa fragment, but not the 33-kDa fragment. The present findings are consistent with the presence of a membrane-binding domain (33 kDa) and an active site domain (38 kDa) in the 70-kDa subunit of PGH synthase. They also suggest that, following cleavage, the 38-kDa fragment retains the structural features responsible for the cyclooxygenase activity and selective aspirin labeling of PGH synthase. PGH synthase undergoes self-catalyzed inactivation by oxidants generated during its catalytic turnover. When PGH synthase, inactivated by treatment with arachidonic acid or hydrogen peroxide, was treated with trypsin it was cleaved two to three times faster than unoxidized enzyme. Addition of heme to oxidized PGH synthase did not reconstitute cyclooxygenase activity or resistance to trypsin cleavage. Spectrophotometric studies demonstrated that oxidatively inactivated enzyme did not bind heme. This implies that oxidation of protein residues as well as the heme prosthetic group is an important determinant of proteolytic sensitivity. Oxidative modification may mark PGH synthase for proteolytic cleavage and turnover.     

Hydrogenase activity of Chlorella vulgaris cells

Cell suspensions of Chlorella vulgaris were found to possess the hydrogenase activity as was confirmed by their ability to absorb H2 in the presence of benzyl viologen, azocarmine and other hydrogen acceptors as well as to produce H2 from reduced methyl viologen. Incubation of the cells in the dark under anaerobic conditions in the atmosphere of H2, N2 or Ar stimulated the activity of hydrogenase and induced its de novo synthesis. Treatment of the cells adapted to anaerobiosis with dry ice or liquid nitrogen considerably increased their hydrogenase activity. The enzyme of the adapted cells was more resistant to the inactivation by O2 and temperature.     

Inhibition and labeling of red beet uridine 5'diphospho-glucose: (1,3)-β-glucan (callose) synthase by chemical modification with formaldehyde and uridine 5'diphospho-pyridoxal

The effects of the lysine-reactive chemical modification reagents, uridine 5’ diphospho (UDP)-pyridoxal and formaldehyde (HCHO), on the activity of membrane-bound and solubilized UDP-Glc: (1,3)-β-D-glucan synthase (callose synthase) from red beet (Beta vulgaris L.) storage tissue were compared. Exposure to micromolar levels of UDP-pyridoxal, or millimolar levels of HCHO in the presence of NaCNBH₃, resulted in complete enzyme inactivation. Conditions for inhibition of membrane-bound enzyme activity by the two reagents were markedly similar; divalent cations were required for inactivation, and complete protection of activity was obtained with EDTA or EGTA. The substrate, UDP-Glc, protected membrane-bound callose synthase against inactivation by UDP-pyridoxal or HCHO, but protected the solubilized enzyme only against inhibition by UDP-pyridoxal, suggesting that the lysine residue modified by both these reagents is at the enzyme active site, and that the site is more open or has a certain conformational flexibility in the solubilized enzyme. Potential UDP-Glc-binding polypeptides of callose synthase were identified by a two-step labeling procedure. First, nonessential lysine residues were blocked by irreversible modification reaction with HCHO or UDP-pyridoxal in the presence of UDP-Glc to protect lysines at UDP-Glc-binding sites. In the second step, proteins were recovered, reacted with [¹⁴C]-HCHO in the absence of UDP-Glc, and polypeptide labeling patterns analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. This procedure reduced incorporation of label by 5- to 8-fold compared to a procedure omitting the preblocking step, and with enzyme partially purified by solubilization in CHAPS followed by product entrapment, labeling was limited to a small set of polypeptides. Taken together with the results of other studies, the data suggest that one or more polypeptides migrating in the 54–57 kDa region are good candidates for the UDP-Glc-binding components of callose synthase.     

Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli

An active tryptic fragment of membrane-bound hydrogenase isoenzyme 2 from anaerobically grown Escherichia coli has been purified. The soluble enzyme derivative was released from the membrane fraction by trypsin cleavage. The purification procedure involved ion-exchange, hydroxyapatite and gel permeation chromatography. The enzyme derivative was purified 100-fold from the membrane fraction and the specific activity of the final preparation was 320 mumol benzyl viologen reduced min-1 mg protein-1 (H2:benzyl viologen oxidoreductase). The native enzyme derivative had an Mr of 180,000 and was composed of equimolar amounts of polypeptides of Mr 61,000 and 30,000. It possessed 12.5 mol Fe, 12.8 mol acid-labile S2- and 3.1 mol Ni/180,000 g enzyme. Antibodies were raised to the purified preparation which cross-reacted with hydrogenase isoenzyme 2 but not with isoenzyme 1 in detergent-dispersed preparations. Western immunoblot analysis revealed that isoenzyme 2 which had not been exposed to trypsin contained cross-reacting polypeptides of Mr 61,000 and 35,000. Trypsin treatment of the membrane-bound enzyme to form the soluble derivative of isoenzyme 2, therefore, cleaves a polypeptide of Mr 35,000 to produce the 30,000-Mr fragment. Trypsin treatment of the detergent-dispersed isoenzyme 2 produces the same fragmentation of the enzyme. Neither of the subunits of the enzyme revealed any immunological identity with those of hydrogenase isoenzyme 1.     

Characterization of a catalase-peroxidase from the hyperthermophilic archaeon Archaeoglobus fulgidus

A putative perA gene from Archaeoglobus fulgidus was cloned and expressed in Escherichia coli BL21(DE3), and the recombinant catalase-peroxidase was purified to homogeneity. The enzyme is a homodimer with a subunit molecular mass of 85 kDa. UV-visible spectroscopic analysis indicated the presence of protoheme IX as a prosthetic group (ferric heme), in a stoichiometry of 0.25 heme per subunit. Electron paramagnetic resonance analysis confirmed the presence of ferric heme and identified the proximal axial ligand as a histidine. The enzyme showed both catalase and peroxidase activity with pH optima of 6.0 and 4.5, respectively. Optimal temperatures of 70 degrees C and 80 degrees C were found for the catalase and peroxidase activity, respectively. The catalase activity strongly exceeded the peroxidase activity, with Vmax values of 9600 and 36 U mg(-1), respectively. Km values for H2O2 of 8.6 and 0.85 mM were found for catalase and peroxidase, respectively. Common heme inhibitors such as cyanide, azide, and hydroxylamine inhibited peroxidase activity. However, unlike all other catalase-peroxidases, the enzyme was also inhibited by 3-amino-1,2,4-triazole. Although the enzyme exhibited a high thermostability, rapid inactivation occurred in the presence of H2O2, with half-life values of less than 1 min. This is the first catalase-peroxidase characterized from a hyperthermophilic microorganism.