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Plasmid pRC41, containing the cyf gene encoding cytochrome c533 from Desulfovibrio vulgaris Hildenborough, was transferred by conjugation from Escherichia coli to Desulfovibrio desulfuricans G200. The structural properties of the purified protein were studied by one-dimensional and two-dimensional NMR. A heterogeneity in the folding of the cytochrome isolated from D. vulgaris Hildenborough and from D. desulfuricans G200 was observed for the oxidized from. Temperature, pH and salt-dependence studies indicated that the heterogeneity does not result from an intermediate in the protein unfolding process, but derives from two conformations which are not in dynamic equilibrium.

It has been shown [Pierik, A. J., Duyvis, M. G., van Helvoort, J. M. L. M., Wolbert, R. B. G. & Hagen, W. R. (1992) Eur. J. Biochem. 205, 111-115] that desulfoviridin, the dissimilatory sulfite reductase of sulfate-reducing bacteria of the genus Desulfovibrio, contains a third, gamma, subunit (11 kDa), in addition to the well-established alpha (50 kDa) and beta (40 kDa) subunits, and an alpha 2 beta 2 gamma 2 subunit structure has been proposed. Cloning and sequencing of the dsvC gene indicated it to encode a protein of 105 amino acids (11.9 kDa; gamma subunit). The finding that the dsvC gene, located on a 3.5-kb SacII fragment, is transcribed in both Escherichia coli and Desulfovibrio vulgaris as an mRNA of only 400-600 nucleotides, and that both the dsvA and dsvB genes are present on a 7.2-kb SacII fragment, indicates that dsvC forms a separate transcriptional unit. The steady-state level of alpha and beta subunits expressed in D. vulgaris Hildenborough cells is rather constant, while that of the gamma subunit increased strongly in the stationary growth phase. Biochemical analysis of the purified protein, expressed in E. coli, and library comparison of its sequence, have so far failed to establish the function of gamma.

Flavodoxin from Desulfovibrio vulgaris (Hildenborough) has been expressed at a high level (3-4% soluble protein) in Escherichia coli by subcloning a minimal insert carrying the gene behind the tac promoter of plasmid pDK6. The recombinant protein was readily isolated and its properties were shown to be identical to those of the wild-type protein obtained directly from D. vulgaris, with the exception that the recombinant protein lacks the N-terminal methionine residue. Detailed measurements of the redox potentials of this flavodoxin are reported for the first time. The redox potential, E2, for the couple oxidized flavodoxin/flavodoxin semiquinone at pH 7.0 is -143 mV (25 degrees C), while the value for the flavodoxin semiquinone/flavodoxin hydroquinone couple (E1) at the same pH is -440 mV. The effects of pH on the observed potentials were examined; E2 varies linearly with pH (slope = -59 mV), while E1 is independent of pH at high pH values, but below pH 7.5 the potential becomes less negative with decreasing pH, indicating a redox-linked protonation of the flavodoxin hydroquinone. D. vulgaris apoflavodoxin binds FMN very tightly, with a value of 0.24 nM for the dissociation constant (Kd) at pH 7.0 and 25 degrees C, similar to that observed with other flavodoxins. In addition, the apoflavodoxin readily binds riboflavin (Kd = 0.72 microM; 50 mM sodium phosphate, pH 7.0, 5 mM EDTA at 25 degrees C) and the complex is spectroscopically very similar to that formed with FMN. The redox potentials for the riboflavin complex were determined at pH 6.5 (E1 = -262 mV, E2 = -193 mV; 25 degrees C) and are discussed in the light of earlier proposals that charge/charge interactions between different parts of the flavin hydroquinone play a crucial role in determining E1 in flavodoxin.

Hydrogenase from Desulfovibrio vulgaris (Hildenborough) is a heterologous dimer of molecular mass 46 + 13.5 kDa. Its two structural genes have been cloned on a 4664-base-pair fragment of known sequence in the vector pUC9. Expression of hydrogenase polypeptides in Escherichia coli transformed with this plasmid is poor (approximately 0.1% w/w of total protein). Deletion of up to 1.9 kb of insert DNA brings the gene encoding for the large subunit in close proximity to the lac promotor of pUC9 and results in a 50-fold increased expression of hydrogenase polypeptides in E. coli. The protein formed is inactive and was purified in order to delineate its defect. Complete purification was achieved with a procedure similar to that used for the isolation of active hydrogenase from D. vulgaris H. The derived protein is also an alpha beta dimer and electron-paramagnetic resonance studies indicate the presence of the electron-transferring ferredoxin-type iron-sulfur clusters. In contrast to the native protein from D. vulgaris H, these can only be reduced with dithionite, not with hydrogen, indicating that the hydrogen-binding active centre which also contains an iron-sulfur cluster is missing.

1. Pyridine nucleotide transhydrogenase of Azotobacter vinelandii purified by affinity chromatography consists of a mixture of polydisperse rods at neutral pH. No other structures are seen by electron microscopy. 2. At high pH (8.5--9.0) the rods depolymerize. Complete depolymerization can be achieved in 0.1 M Tris-Cl pH 9.0. The depolymerized enzyme has a molecular weight of 421000 (sedimentation equilibrium), its sedimentation coefficient s20, w = 15 S and its Stokes' radius Rs = 7 nm. Since gel electrophoresis in the presence of sodium dodecyl sulphate shows that transhydrogenase consists of a single polypeptide chain of molecular weight (54 +/- 2) X 10(3) it follows that the depolymerized enzyme has an octameric quaternary structure. We propose that this octamer serves as the functional monomeric unit ('unimer') from which the polymeric form of transhydrogenase is constructed. 3. Gel filtration and sucrose gradient centrifugation studies of cell-free extracts from A. vinelandii show the unimer to be the predominant active species.

1. Pyridine nucleotide transhydrogenase from Azotobacter vinelandii was purified with a scaled-up procedure. In a typical purification 500 ml cell-free extract from 200 g cells is loaded on an Ado-2',5'-P2--Sepharose 4B affinity column (20 ml bed volume). After washing, the enzyme is desorbed with 2'AMP at neutral pH and further purified by Sephadex G-200 gel chromatography. The enzyme (10--12 mg) is obtained in 40--60% yield and is homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. 2. The homogeneity of the purified enzyme is also apparent from electron microscopy studies, where the enzyme appears as a polydisperse set of polymers without contaminating structures and from fluorescence lifetime studies by the method of single-photon counting. The flavin fluorescence appears to decay with a single lifetime tau = 2.5 ns. The polymeric nature of transhydrogenase can be aptly demonstrated by density gradient centrifugation in the presence of KBr. After centrifuging for 50 h at 160 000 X g and 10 degrees C the enzyme is concentrated in a narrow fluorescent band with buoyant density rho b = 1.305 g cm-3. 3. The arrangement of subunits in the transhydrogenase polymer has been derived from optical diffraction studies of electron micrographs. The polymers are built up from a linear assembly of tetramers. Four subunits are placed in a rhomb with sides of 13.5 mm and an angle of 45 degrees (135 degrees) between the sides. A second tetramer is located staggered on top of the first one. Since a variety of other studies have indicated that the polymers dissociate into octamers under alkaline conditions [Voordouw, G. et al. (1979 Eur. J. Biochem. 98,447--454] we conclude that this smallest functional unit is build up from two tetramers.

Two types of pyridine nucleotide transhydrogenases have been reported in living organisms. The energy-linked transhydrogenase is found in mitochondria and in certain heterotrophic and photosynthesizing bacteria, while the non-energy-linked transhydrogenase is found in certain heterotrophic bacteria. The presence of a structurally similar non-energy-linked transhydrogenase in Azotobacter vinelandii, Pseudomonas aeruginosa and Pseudomonas fluorescens is readily shown in extracts from these bacteria with Western (protein) blotting. This non-energy-linked enzyme is lacking in Escherichia coli, while the presence of a structurally similar energy-linked enzyme in E. coli and in beef heart mitochondria is indicated with the Western blotting technique. Spinach (Spinacia oleracea) lacks the non-energy-linked transhydrogenase occurring in bacteria. The chloroplast enzyme ferredoxin:NADP+ oxidoreductase, which exhibits non-energy-linked transhydrogenase activity, is immunologically distinct from the bacterial transhydrogenases. In order to provide a rationale for the distribution of the two types of pyridine nucleotide transhydrogenases, the steady-state degrees of reduction of the NADP(H) and NAD(H) pools in A. vinelandii (R'NADP(H) and R'NAD(H)) have been measured for cells metabolizing sucrose at a variable oxygen flux (phi O2). It is found that the degree of reduction of the NADP(H) pool is always higher than that of the NAD(H) pool (R'NADP(H) greater than R'NAD(H)) except when phi O2 goes to zero (R'NADP(H) approximately equal to R'NAD(H)). Comparison of these results with literature values indicates that the inequality R'NADP(H) greater than R'NAD(H) is always found in a membrane-enclosed compartment, irrespective of the type of transhydrogenase present. This allows an understanding of the function of the two types of pyridine nucleotide transhydrogenases in vivo. The physiological role of non-energy-linked transhydrogenase is to catalyze the reaction NADPH + NAD+ leads to NADP+ + NADH, that of energy-linked transhydrogenase to catalyze the reaction NADH + NADP+ leads to NADPH + NAD+. Since at equilibrium R'NADP(H) approximately equal to R'NAD(H) the inequality R'NADP(H) greater than R'NAD(H) under steady-state conditions explains the energy requirement in the latter reaction. The dependence of the non-energy-linked transhydrogenase activity of ferredoxin:NADP+ oxidoreductase on R'NADP(H) is compared with that of A, vinelandii transhydrogenase. The results indicate that this activity is unlikely to be of physiological importance in plant chloroplasts.

The dissociation of D-ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach, which consists of eight large subunits (L, 53 kDa) and eight small subunits (S, 14 kDa) and thus has a quarternary structure L8S8, has been investigated using a variety of physical techniques. Gel chromatography using Sephadex G-100 indicates the quantitative dissociation of the small subunit S from the complex at 3-4 M urea (50 mM Tris/Cl pH 8.0, 0.5 mM EDTA, 1 mM dithiothreitol and 5 mM 2-mercaptoethanol). The dissociated S is monomeric. Analytical ultracentrifuge studies show that the core of large subunits, L, remaining at 3-4 M urea sediments with S20, w = 15.0 S, whereas the intact enzyme (L8S8) sediments with S20, w = 17.7S. The observed value is consistent with a quarternary structure L8. The dissociation reaction in 3-4 M urea can thus be represented by L8S8----L8 + 8S. At urea concentrations c greater than 5 M the L8 core dissociates into monomeric, unfolded large subunits. A large decrease in fluorescence emission intensity accompanies the dissociation of the small subunit S. This change is completed at 4 M urea. No changes are observed upon dissociating the L8 core. The kinetics of dissociation of the small subunit, as monitored by fluorescence spectroscopy, closely follow the kinetics of loss of carboxylase activity of the enzyme. Studies of the circular dichroism of D-ribulose-1,5-bisphosphate carboxylase in the wavelength region 200-260 nm indicate two conformational transitions. The first one ([0]220 from -8000 to -3500 deg cm2 dmol-1) is completed at 4 M urea and corresponds to the dissociation of the small subunit and coupled conformational changes. The second one ([0]220 from -3500 to -1200 deg cm2 dmol-1) is completed at 6 M urea and reflects the dissociation and unfolding of large subunits from the core. The effect of activation of the enzyme by addition of MgCl2 (10 mM) and NaHCO3 (10 mM) on these conformational transitions was investigated. The first conformational transition is then shifted to higher urea concentrations: a single transition ([0]220 from -8000 to -1200 deg cm2 dmol-1) is observed for the activated enzyme. From the urea dissociation experiments we conclude that both large (L) and small (S) subunits are important for carboxylase activity of spinach D-ribulose-1,5-bisphosphate carboxylase: the L-S subunit interactions tighten upon activation and dissociation of S leads to a coupled, proportional loss of enzyme activity.

The nucleotide sequence of the 4.7-kb SalI/EcoRI insert of plasmid pHV 15 containing the hydrogenase gene from Desulfovibrio vulgaris (Hildenborough) has been determined with the dideoxy chain-termination method. The structural gene for hydrogenase encodes a protein product of molecular mass 45820 Da. The NH2-terminal sequence of the enzyme deduced from the nucleic acid sequence corresponds exactly to the amino acid sequence determined by Edman degradation. The nucleic acid sequence indicates that a N-formylmethionine residue precedes the NH2-terminal amino acid Ser-1. There is no evidence for a leader sequence. The NH2-terminal part of the hydrogenase shows homology to the bacterial [8Fe-8S] ferredoxins. The sequence Cys-Ile-Xaa-Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Cys-Pro-Xaa-Xaa-Ala-(Ile) occurs twice both in the hydrogenase and in [8Fe-8S] ferredoxins, where the Cys residues have been shown to coordinate two [4Fe-4S] clusters [Adman, E. T., Sieker, L. C. and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987-3996]. These results, therefore, suggest that two electron-transferring ferredoxin-like [4Fe-4S] clusters are located in the NH2-terminal segment of the hydrogenase molecule. There are ten more Cys residues but it is not clear which four of these could participate in the formation of the third cluster, which is thought to be the hydrogen binding centre. Another gene, encoding a protein of molecular mass 13493 Da, was found immediately downstream from the gene for the 46-kDa hydrogenase. The nucleic acid sequence suggests that the hydrogenase and the 13.5-kDa protein belong to a single operon and are coordinately expressed. Since dodecylsulfate gel electrophoresis of purified hydrogenase indicates the presence of a 13.5-kDa polypeptide in addition to the 46-kDa component, it is proposed that the hydrogenase from D. vulgaris (Hildenborough) is a two-subunit enzyme.

Using oligonucleotide-directed mutagenesis of the gene encoding the small subunit (rbcS) from Anacystis nidulans mutant enzymes have been generated with either Trp-54 of the small subunit replaced by a Phe residue, or with Trp-57 replaced by a Phe residue, whereas both Trp-54 and Trp-57 have been replaced by Phe residues in a double mutant. Trp-54 and Trp-57 are conserved in all amino acid sequences or the small subunit (S) that are known at present. The wild-type and mutant forms of Rubisco have all been purified to homogeneity. The wild-type enzyme, purified from Escherichia coli is indistinguishable from enzyme similarly purified from A. nidulans in subunit composition, subunit molecular mass and kinetic parameters (Vmax CO2 = 2.9 U/mg, Km CO2 = 155 microM). The single Trp mutants are indistinguishable from the wild-type enzyme by criteria (a) and (b). However, whereas, Km CO2 is also unchanged, Vmax CO2 is 2.5-fold smaller than the value for the wild-type enzyme for both mutants, demonstrating for the first time that single amino acid replacements in the non-catalytic small subunit influence the catalytic rate of the enzyme. The specificity factor tau, which measures the partitioning of the active site between the carboxylase and oxygenase reactions, was found to be invariant. Since tau is not affected by these mutations we conclude that S is an activating not a regulating subunit.

The purification procedure for p-hydroxybenzoate hydroxylase has been modified by replacement of the DEAE-cellulose (DE-32) column in the original procedure by a Sephadex--Cibacron-blue affinity column. In this way the yield of enzyme could be improved from 16% to about 40--50%. Preparative gel chromatography indicated that the enzyme does not exist as a monomeric species as earlier believed but mainly as a dimer. Sodium dodecyl sulfate gel electrophoresis of purified enzyme revealed a minimum relative molecular mass (Mr) of 43000--45000. Analytical gel chromatography, sedimentation equilibrium and sedimentation velocity experiments showed that the enzyme exists in solution mainly as a dimer but also in higher-order quaternary structures (presumably tetramer and hexamer). Temperature dependence of the distribution of the oligomers suggests that the association is of hydrophobic nature. The amino acid composition of the enzyme is also presented. The enzyme contains no disulfide but five sulfhydryl groups. In the native state of the enzyme only one sulfhydryl group is accessible to N-ethylmaleimide or 5,5'-dithiobis(2-nitrobenzoic acid). The iso-electric point of the enzyme was found to be 5.8.

The gene encoding the redox protein cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) has been cloned using two synthetic oligonucleotides (one 17-mer and one 18-mer), designed to recognize the structural gene. Plasmid pCYC3 was derived from the clone and contains a 7.5 X 10(3)-base EcoRI-HindIII insert of D. vulgaris DNA in pUC9. A 674-base-pair fragment of this insert was sequenced with the dideoxy-chain-termination procedure and found to contain the entire structural gene encoding cytochrome c3 bracketed by apparent Escherichia coli consensus sequences for initiation and termination of transcription. The amino acid sequence of 107 residues, derived from protein sequencing [Trousil, E. B. and Campbell, L. L. (1974) J. Biol. Chem. 249, 386-393], is confirmed by the nucleic acid sequence, which shows in addition that it is preceded by a hydrophobic, positively charged signal sequence of 21 residues. This amino-terminal extension functions in the export of cytochrome c3, which is thought to reside in the periplasm of D. vulgaris.

1. Purified pyridine nucleotide transhydrogenase from Azotobacter vinelandii contains three thiol residues as judged by titration with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions. 2. In the native conformation of the transhydrogenase only a single thiol residue is titrated. Modification of this exposed thiol does not influence transhydrogenase activity. 3. The two less exposed thiol residues can be reacted in part with either p-chloromercuribenzoate or N-ethyl-maleimide. Modification of one residue leads to loss of 40-60% of the enzyme activity in both the forward (NAD+ + NADPH leads to NADH + NADP+) and reverse reaction. The strong inhibitory action of phosphate ions on the reverse reaction [Voordouw et al. (1980) Eur. J. Biochem. 107, 337-344] is abolished after treatment with p-chloromercuribenzoate. Reaction with phenylmercurichloride or p-aminophenylmercuriacetate causes a similar activity loss without affecting the inhibitory action of phosphate. 4. The interaction of the divalent thiol inhibitor p-aminophenylarsenoxide with transhydrogenase was found to be reversible and is characterized by an association constant of 6.3 x 10(5) M-1 at 25 degrees C in 50 mM sodium phosphate pH 7.50. This reversibility indicates formation of a cyclic dithiolarsinite derivative with considerable ring strain. The activity of p-aminophenylarsenoxide-transhydrogenase is modulated by phosphate and magnesium ions. The activity of the transhydrogenase . p-aminophenylarsenoxide complex in the forward reaction is inhibited by phosphate and stimulated by magnesium ions. The reverse reaction is not catalyzed by the enzyme-inhibitor complex. 5. The presence of an activity modulating site in transhydrogenase which binds phosphate ions and has the two less exposed thiol residues in close proximity is indicated by the results.

The gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough) has been cloned in Escherichia coli. D. vulgaris DNA was digested with the restriction endonucleases EcoRI and SalI and ligated into the vector pUC9 [Vieira, J. & Messing, J. (1982) Gene 19, 259-268], which had been cut with these same enzymes. Approximately 9000 recombinant clones were obtained by transformation of E. coli JM 101 followed by growth on rich plates with ampicillin for selection and isopropyl-beta-D-thiogalactoside and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside present for detection of recombinants. The recombinant clones were then screened for production of immunoreactive proteins with rabbit antisera against purified hydrogenase and 125I-labelled protein A. 28 positive clones were found in this initial screening. These were further tested in an immunocompetition experiment, which showed that the protein product from one clone behaved identically to purified hydrogenase. The plasmid pHV15 isolated from this clone has a 4.7 X 10(3)-base-pair SalI/EcoRI insert. Cells of E. coli JM 101 transformed with pHV 15 produce a hydrogenase polypeptide of molecular mass 46 kDa as detected by Western blotting. The mass, as well as the Cleveland mapping pattern of the polypeptide produced by E. coli, are identical with those of the hydrogenase isolated from D. vulgaris (Hildenborough). Southern blotting of restriction-enzyme-digested D. vulgaris DNA, using the nick-translated 4.7 X 10(3)-base-pair SalI/EcoRI fragment as a probe, indicates the presence of a single gene with an internal PstI site. The NH2-terminal sequence of the hydrogenase was determined to be: (sequence in text). This information should allow an unambiguous identification of the hydrogenase gene.

The Mo-Fe protein of nitrogenase from both Azotobacter vinelandii and Klebsiella pneumoniae (Av1 and Kp1, respectively) consists of four subunits of similar, but not identical, relative molecular mass. The hydrodynamic properties of Av1 (sedimentation and diffusion coefficient) and its total relative molecular mass are very similar to those of Kp1 and catalase from bovine liver, a tetramer of four identical subunits. By electron microscopy the Av1, Kp1 and catalase tetramers are seen as protein particles of diameter 9.0-10.0 nm; no details of the subunit structure can be observed. Av1 (but not Kp1) forms regular polymers of variable length at low ionic strength in the presence of MgCl2. The structure of these polymers, of diameter 21.2 nm, is complex. Optical diffraction studies give a smallest repeating distance of 8.4 nm (corresponding to the diameter of the Av1 tetramer) and indicate a four-start helix. The latter structure is incompatible with a flat, square subunit arrangement of the Av1 tetramer as proposed by Stasny et al. [(1974) J. Cell. Biol. 60, 311-316]. We propose, therefore, that the subunit arrangement of the Av1 tetramer is of the tetrahedral type. This has also been proposed for the catalase tetramer from optical diffraction studies of electron micrographs of catalase tubes indicating a 222 symmetry [Kiselev, D. A., De Rosier, N. J. and Klug, A. (1968) J. Mol. Biol. 35, 561-566]. Our proposal is in agreement with the recent finding that Av1 protein crystals belong to the P2(1) space group [Weiniger, M. S. and Mortenson, L. E. (1982) Proc. Natl Acad Sci. USA, 79, 378-380].