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.

ABSTRACT 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 NH 4 + 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 NH 4 + 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 .

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 Cred1; (ii) uncoupled states at <−320 mV in the absence of CO2 referred to as Cunc; and (iii) another spin-coupled state at <−320 mV in the presence of CO2 that gives rise to an EPR signal termed Cred2B. Because there is no initial CODH activity at potentials that give rise to Cred1, the state (Cred1) 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.

10.1128/JB.183.5.1610-1620.2001. 2001, 183(5):1610. DOI: J. Bacteriol. W. Ludden and Gary P. Roberts Yaoping Zhang, Edward L. Pohlmann, Cale M. Halbleib, Paul pneumoniae Klebsiella Regulatory System in Reductase-Activating Glycohydrolase Dinitrogenase − Transferase Dinitrogenase Reductase ADP-Ribosyl Rhodospirillum rubrum Expression of the Dinitrogenase Reductase by Heterologous Reversible ADP-Ribosylation of and Its Homolog GlnK on II Effect of P

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 [Fe4S4](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 [Fe4S4] cluster from R, rubrum Fe protein. DRAT was found to have a specificity opposite that of DRAG, exhibiting low (<2%) 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 mw NH4Cl 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.

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.

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.

ABSTRACT 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.

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 (EC 50 ) of 5.0 ± 0.5 mM; guanosine disphosphate had an EC 50 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.

Biological nitrogen fixation represents a major energy drain for the cell and it is therefore not surprising that organisms have developed a range of mechanisms for the transcriptional and post-transcriptional regulation of that process. One of the best understood post-transcriptional regulatory mechanisms is reversible ADP-ribosylation, which has been found in some phototrophs and the Azospirillum genus.

The regulation of dinitrogenase reductase by ADP-ribosylation is depicted in Fig. 1 and is described in detail in a review.1 The ADP-ribose moiety of P-NAD+ is transferred to arginine-101 of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT, the draT gene product).2.3 Modification of one of the two identical subunits of dinitrogenase reductase renders the protein inactive4 and modification of both subunits of the same dimer has not been observed. The structure of dinitrogenase reductase shows arg-101 to be at the surface of the protein near the FeS center.5 ADP-ribose is attached to arginine-101 by an a-specific bond to the guanidinium N.6.7 The ADP-ribosylated dinitrogenase reductase in its reduced, native form can be activated by dinitrogenase reductase activating glycohydrolase (DRAG, the draG gene product) in an MgATP- and Mn2+-dependent reaction. DRAG removes ADP-ribose and regenerates the site of modification in the process.8 ADP-ribose can be removed from both oxidized and oxygen-denatured dinitrogenase reductase in the absence of MgATP, but a divalent metal (Mn2+) is still required.910 Likewise, DRAG can hydrolyze low molecular weight substrates such as dansyl arginine methylester ADP-ribose in the absence of nucleotide.9 DRAT is unable to ADPribosylate small molecule substrates.3

Nitrogenase activity in Rhodospirillum rubrum, Azospirillum brasilense and related bacteria is regulated by the reversible ADP-ribosylation of arg-101 of the dinitrogenase reductase protein (Ludden and Roberts 1989). Only one of the two arg-101 residues of the NifH homodimer is modified at one time, presumably because of steric hindrance. The ADP-ribosylation of dinitrogenase reductase prevents its productive association with dinitrogenase and thus neither electron transfer nor ATP hydrolysis occurs (Murrell et al. 1988). The NAD-dependent ADP-ribosylation of dinitrogenase reductase is catalyzed by DRAT (Dinitrogenase Reductase ADP-ribosyl Transferase) and the removal of ADP-ribose is catalyzed by DRAG (Dinitrogenase Reductase Activating Glycohydrolase). DRAT and DRAG are encoded by the draTG operon, which is not co-regulated with the nif genes but which is found near the nifHDK operon in R. rubrum, A. lipoferum and A. brasilense (Fitzmaurice et al. 1989; Fu et al. 1989; Fu et al. 1990; Zhang et al. 1992). Both DRAT and DRAG activities are regulated in vivo. Regulation of DRAG was established when 32Plabelled ADP-ribose could not be chased from dinitrogenase reductase during conditions favoring modification (Kanemoto and Ludden 1984). If DRAG was unregulated, the labelled ADP-ribose group would have been continuously replaced (chased) by unlabelled ADP-ribose. tDRAT is regulated in vivo becuase a mutant lacking DRAG but containing DRAT activity accumulated dinitrogenase in the active form until a signal (darkness or ammonium) was given to initiate the modification (Liang et al. 1991). The regulation of both DRAT and DRAG is through inhibition of their activities by other factors, because both enzymes are active in the purified form and require no other factors for activation.