Redox-dependent activation of CO dehydrogenase from Rhodospirillum rubrum.

Studies of initial activities of carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum show that CODH is mostly inactive at redox potentials higher than -300 mV. Initial activities measured at a wide range of redox potentials (0--500 mV) fit a function corresponding to the Nernst equation with a midpoint potential of -316 mV. Previously, extensive EPR studies of CODH have suggested that CODH has three distinct redox states: (i) a spin-coupled state at -60 to -300 mV that gives rise to an EPR signal termed C(red1); (ii) uncoupled states at <-320 mV in the absence of CO(2) referred to as C(unc); and (iii) another spin-coupled state at <-320 mV in the presence of CO(2) that gives rise to an EPR signal termed C(red2B). Because there is no initial CODH activity at potentials that give rise to C(red1), the state (C(red1)) is not involved in the catalytic mechanism of this enzyme. At potentials more positive than -380 mV, CODH recovers its full activity over time when incubated with CO. This reductant-dependent conversion of CODH from an inactive to an active form is referred to hereafter as "autocatalysis." Analyses of the autocatalytic activation process of CODH suggest that the autocatalysis is initiated by a small fraction of activated CODH; the small fraction of active CODH catalyzes CO oxidation and consequently lowers the redox potential of the assay system. This process is accelerated with time because of accumulation of the active enzyme.

Effect of P(II) and its homolog GlnK on reversible ADP-ribosylation of dinitrogenase reductase by heterologous expression of the Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyl transferase-dinitrogenase reductase-activating glycohydrolase regulatory system in Klebsiella pneumoniae.

Reversible ADP-ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADP-ribosyl transferase-dinitrogenase reductase-activating glycohydrolase (DRAT-DRAG) regulatory system, has been characterized in Rhodospirillum rubrum and other nitrogen-fixing bacteria. To investigate the mechanisms for the regulation of DRAT and DRAG activities, we studied the heterologous expression of R. rubrum draTG in Klebsiella pneumoniae glnB and glnK mutants. In K. pneumoniae wild type, the regulation of both DRAT and DRAG activity appears to be comparable to that seen in R. rubrum. However, the regulation of both DRAT and DRAG activities is altered in a glnB background. Some DRAT escapes regulation and becomes active under N-limiting conditions. The regulation of DRAG activity is also altered in a glnB mutant, with DRAG being inactivated more slowly in response to NH4+ treatment than is seen in wild type, resulting in a high residual nitrogenase activity. In a glnK background, the regulation of DRAT activity is similar to that seen in wild type. However, the regulation of DRAG activity is completely abolished in the glnK mutant; DRAG remains active even after NH4+ addition, so there is no loss of nitrogenase activity. The results with this heterologous expression system have implications for DRAT-DRAG regulation in R. rubrum.

Evidence for a ligand CO that is required for catalytic activity of CO dehydrogenase from Rhodospirillum rubrum.

Radiolabeling studies support the existence of a nonsubstrate CO ligand (CO(L)) to the Fe atom of the proposed [FeNi] cluster of carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum. Purified CODH has variable amounts of CO(L) dissociated depending on the extent of handling of the proteins. This dissociated CO(L) can be restored by incubation of CODH with CO, resulting in a 30-40% increase in initial activity relative to as-isolated purified CODH. A similar amount of CO(L) binding is observed when as-isolated purified CODH is incubated with (14)CO: approximately 0.33 mol of CO binds per 1 mol of CODH. Approximately 1 mol of CO was released from CO-preincubated CODH upon denaturation of the protein. No CO could be detected upon denaturation of CODH that had been incubated with cyanide. CO(L) binds to both Ni-containing and Ni-deficient CODH, indicating that CO(L) is liganded to the Fe atom of the proposed [FeNi] center. Furthermore, the Ni in the CO(L)-deficient CODH can be removed by treatment with a Ni-specific chelator, dimethylglyoxime. CO preincubation protects the dimethylglyoxime-labile Ni, indicating that CO(L) is also involved in the stability of Ni in the proposed [FeNi] center.

Effects of perturbations of the nitrogenase electron transfer chain on reversible ADP-ribosylation of nitrogenase Fe protein in Klebsiella pneumoniae strains bearing the Rhodospirillum rubrum dra operon.

The redox state of nitrogenase Fe protein is shown to affect regulation of ADP-ribosylation in Klebsiella pneumoniae strains transformed by plasmids carrying dra genes from Rhodospirillum rubrum. The dra operon encodes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase, enzymes responsible for the reversible inactivation, via ADP-ribosylation, of nitrogenase Fe protein in R. rubrum. In bacteria containing the dra operon in their chromosomes, inactivation occurs in response to energy limitation or nitrogen sufficiency. The dra gene products, expressed at a low level in K. pneumoniae, enable transformants to reversibly ADP-ribosylate nitrogenase Fe protein in response to the presence of fixed nitrogen. The activities of both regulatory enzymes are regulated in vivo as described in R. rubrum. Genetic perturbations of the nitrogenase electron transport chain were found to affect the rate of inactivation of Fe protein. Strains lacking the electron donors to Fe protein (NifF or NifJ) were found to inactivate Fe protein more quickly than a strain with wild-type background. Deletion of nifD, which encodes a subunit of nitrogenase MoFe protein, was found to result in a slower inactivation response. No variation was found in the reactivation responses of these strains. It is concluded that the redox state of the Fe protein contributes to the regulation of the ADP-ribosylation of Fe protein.

Regulation of biological nitrogen fixation.

Biological nitrogen fixation, a process found only in some prokaryotes, is catalyzed by the nitrogenase enzyme complex. Bacteria containing nitrogenase occupy an indispensable ecological niche, supplying fixed nitrogen to the global nitrogen cycle. Due to this inceptive role in the nitrogen cycle, diazotrophs are present in virtually all ecosystems, with representatives in environments as varied as aerobic soils (e.g., Azotobacter species), the ocean surface layer (Trichodesmium) and specialized nodules in legume roots (Rhizobium). In any ecosystem, diazotrophs must respond to varied environmental conditions to regulate the tremendously taxing nitrogen fixation process. All characterized diazotrophs regulate nitrogenase at the transcriptional level. A smaller set also possesses a fast-acting post-translational regulation system. Although there is little apparent variation in the sequences and structures of nitrogenases, there appear to be almost as many nitrogenase-regulating schemes as there are nitrogen-fixing species. Herein are described the paradigms of nitrogenase function, transcriptional control and post-translational regulation, as well as the variations on these schemes, described in various nitrogen-fixing bacteria. Regulation is described on a molecular basis, focusing on the functional and structural characteristics of the proteins responsible for control of nitrogen fixation.

Regulation of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase by a redox-dependent conformational change of nitrogenase Fe protein.

The nitrogenase-regulating enzymes dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase-activating glycohydrolase (DRAG), from Rhodospirillum rubrum, were shown to be sensitive to the redox status of the [Fe(4)S(4)](1+/2+) cluster of nitrogenase Fe protein from R. rubrum or Azotobacter vinelandii. DRAG had <2% activity with oxidized R. rubrum Fe protein relative to activity with reduced Fe protein. The activity of DRAG with oxygen-denatured Fe protein or a low molecular weight substrate, N(alpha)-dansyl-N(omega)-(1,N(6)-etheno-ADP-ribosyl)-arginine methyl ester, was independent of redox potential. The redox midpoint potential of DRAG activation of Fe protein was -430 mV versus standard hydrogen electrode, coinciding with the midpoint potential of the [Fe(4)S(4)] cluster from R. rubrum Fe protein. DRAT was found to have a specificity opposite that of DRAG, exhibiting low (<20%) activity with 87% reduced R. rubrum Fe protein relative to activity with fully oxidized Fe protein. A mutant of R. rubrum in which the rate of oxidation of Fe protein was substantially decreased had a markedly slower rate of ADP-ribosylation in vivo in response to 10 mM NH(4)Cl or darkness stimulus. It is concluded that the redox state of Fe protein plays a significant role in regulation of the activities of DRAT and DRAG in vivo.

Characterization of the interaction of dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum with bacterial membranes.

The interaction of dinitrogenase reductase-activating glycohydrolase (DRAG) with bacterial membranes and the solubilization of DRAG in response to nucleotides were characterized. Purified DRAG from Rhodospirillum rubrum reversibly bound bacterial pellet fractions from Rsp. rubrum and other nitrogen-fixing bacteria. DRAG saturated the membrane fraction of Rsp. rubrum at a concentration of 0.2 mol DRAG/mol bacteriochlorophyll, suggesting that the DRAG-binding species is prevalent in the membrane. DRAG bound poorly to phospholipid vesicles, suggesting a protein requirement for DRAG interaction with the membrane. Guanosine and uridine tri- and di-nucleotides specifically dissociated DRAG from the pellet fractions of Rsp. rubrum and Azotobacter vinelandii, while adenosine nucleotides had no dissociative effect. Guanosine 5'-triphosphate dissociated DRAG from the membrane at a concentration causing 50% dissociation (EC50) of 5.0 +/- 0.5 mM; guanosine disphosphate had an EC50 of 15.0 +/- 2.0 mM. We propose that GTP is a potential participant in the regulation of DRAG, possibly controlling the extent of DRAG association with the membrane. Keywords Rhodospirillum rubrum. Membrane. association. Nitrogenase regulation. Nucleotide bindinghttp://link.springer-ny. com/link/service/journals/00203/bibs/172n1p51.html