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.

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 reductantdependent 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.C arbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum is a nickel-iron-sulfur enzyme that carries out the reversible oxidation of CO to CO 2 (1-4). Kinetic analyses have demonstrated that Ni is required for CODH activity (3,5,6). Previous spectroscopic studies have indicated that CODH contains the following iron-sulfur clusters: (i) a B-cluster with properties typical for an all-cysteinyl-liganded [Fe 4 S 4 ] cluster; and (ii) an Ni-containing C-cluster that we have proposed to contain an [FeNi] binuclear subcluster (proposed subcluster of C-cluster) component bridged to or interacting with a slowrelaxing (EPR properties) Fe-containing subcluster component hereafter referred to as FeS C (7). Previously, the C-cluster was thought to contain a single Ni atom bridged to an [Fe 4 S 4 ] cluster (8). The B-cluster is thought to mediate electron transfer from the C-cluster to an electron acceptor, CooF (9, 10). EPR spectroscopic studies of CODH from R. rubrum have revealed that the two C-subclusters ([FeNi] (8,10). In addition, CODH contains a third cluster, the D-cluster. This cluster is an all-cysteinyl-liganded Fe 4 S 4 cluster that bridges the two subunits of the CODH dimer (data not shown). The redox events and role of this cluster are not known at this time.The redox-dependent EPR signals of the metal clusters and the corresponding spin states are suggested to be (10) (i) at the potential range of Ϫ60 to Ϫ300 mV, a g ave ϭ 1.87 signal is observed and is termed C red1 . The C red1 EPR signal is suggested to result from a spin coupling of [Fe 3ϩ The measurement of C...

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.

Reversible ADP-ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADPribosyl 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.Biological nitrogen fixation, the conversion of atmospheric nitrogen to ammonium, is catalyzed by the nitrogenase complex, which consists of two proteins: dinitrogenase (or MoFe protein) and dinitrogenase reductase (or Fe protein) (7). It is a very energy-demanding process and is thus tightly regulated at both transcriptional and posttranslational levels.Transcriptional regulation of the nif genes has been found in all studied nitrogen-fixing bacteria and is best characterized in Klebsiella pneumoniae, a free-living nitrogen-fixing bacterium, where it involves the general nitrogen regulation (ntr) system (36). Analysis of the ntr regulatory system in K. pneumoniae and Escherichia coli (36, 39) has shown that it controls the transcription of many genes involved in nitrogen fixation and assimilation, such as glnA (encoding glutamine synthetase [GS]) and nifA (encoding the transcriptional activator for the other nif genes). The ntr system involves a number of gene products, including those of glnD, ntrA, ntrB, ntrC, and glnB, glnD encodes a bifunctional, uridylyltransferase-uridylyl-removing enzyme (UTase-UR) that is believed to be the sensor of the intracellular concentration of glutamine in the cell. UTase-UR reversibly controls the activity of the P II protein (the gene product of glnB) by uridylylation or deuridylylation. P II is responsible for sensing ␣-ketoglutarate (␣-KG) in E. coli (24), and it controls NtrB (NRII) activity. NtrB and NtrC (the gene products of ntrB and ntrC) belong to the family of twocomponent regulators. NtrB is a histidine kinase that phosphorylates NtrC (NRI) under nitrogen-limiting conditions and also can act as a phosphatase to dephosphorylate NtrC unde...

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