Paul W. Ludden

Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

The iron-molybdenum cofactor (FeMo-co), located at the active site of the molybdenum nitrogenase, is one of the most complex metal cofactors known to date. During the past several years, an intensive effort has been made to purify the proteins involved in FeMo-co synthesis and incorporation into nitrogenase. This effort is starting to provide insights into the structures of the FeMo-co biosynthetic intermediates and into the biochemical details of FeMo-co synthesis.

Life on carbon monoxide: X-ray structure of Rhodospirillum rubrum Ni-Fe-S carbon monoxide dehydrogenase

A crystal structure of the anaerobic Ni-Fe-S carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum has been determined to 2.8-Å resolution. The CODH family, for which the R. rubrum enzyme is the prototype, catalyzes the biological oxidation of CO at an unusual Ni-Fe-S cluster called the C-cluster. The Ni-Fe-S C-cluster contains a mononuclear site and a four-metal cubane. Surprisingly, anomalous dispersion data suggest that the mononuclear site contains Fe and not Ni, and the four-metal cubane has the form [NiFe3S4] and not [Fe4S4]. The mononuclear site and the four-metal cluster are bridged by means of Cys531 and one of the sulfides of the cube. CODH is organized as a dimer with a previously unidentified [Fe4S4] cluster bridging the two subunits. Each monomer is comprised of three domains: a helical domain at the N terminus, an α/β (Rossmann-like) domain in the middle, and an α/β (Rossmann-like) domain at the C terminus. The helical domain contributes ligands to the bridging [Fe4S4] cluster and another [Fe4S4] cluster, the B-cluster, which is involved in electron transfer. The two Rossmann domains contribute ligands to the active site C-cluster. This x-ray structure provides insight into the mechanism of biological CO oxidation and has broader significance for the roles of Ni and Fe in biological systems.

Maturation of nitrogenase: a biochemical puzzle.

The nitrogenase enzyme catalyzes the reductive breakage of the very strong triple bond of N2 to generate NH3 in a process known as biological nitrogen fixation. Biological nitrogen fixation is an essential step in the nitrogen cycle in the biosphere, and it is a major contributor to the nitrogen

Regulation of Nitrogenase Activity by Reversible ADP Ribosylation

Publisher Summary This chapter discusses the molecular bases for Gest and Kamens observations and attempts to put these observations into perspective with respect to the regulation of the nitrogenase enzyme complex by the reversible ADP ribosylation of dinitrogenase reductase. Although it is often difficult to demarcate where to begin a literature review, in this case the relevant literature begins precisely with the publication of consecutive papers by Gest and Kamen in Science in 1949. In these two brief papers, Gest and Kamen described their observations that the photosynthetic bacterium Rhodospirillum rubrum fixes nitrogen, that this activity is light-dependent, and that the activity is inhibited by ammonia. Thereafter, the discovery of dinitrogenase reductase-activating glycohydrolase (DRAG) allowed the purification and characterization of the dinitrogenase reductase from R. rubrum in its inactive form. The purification of the protein could be monitored by assaying the enzyme activity after activation. DRAG was purified based on its ability to activate ADP-ribosylated dinitrogenase reductase, even though it was not known at the time that ADP-ribose was the modifying group. The enzyme was found to be associated with the membrane fraction of extracts. Consequently, it was observed that Mono-ADP ribosylation exhibited a mechanism of action of the diphtheria toxin. This suggested that ADP ribosylation might play a role in normal cell metabolism as well by regulating the activity of key enzymes. The R. rubrum nitrogenase system provides an excellent experimental system for the investigation of such regulation.

Genes coding for the reversible ADP-ribosylation system of dinitrogenase reductase from Rhodospirillum rubrum

SummaryNitrogen fixation activity in the photosynthetic bacterium Rhodospirillum rubrum is controlled by the reversible ADP-ribosylation of the dinitrogenase reductase component of the nitrogenase enzyme complex. This report describes the cloning and characterization of the genes encoding the ADP-ribosyltransferase (draT) and the ADP-ribosylglycohydrolase (draG) involved in this regulation. These genes are shown to be contiguous on the R. rubrum chromosome and highly linked to the nifHDK genes. Sequence analysis revealed the use of TTG as the initiation codon of the draT gene as well as a potential open reading frame immediately downstream of draG. The mono-ADP-ribosylation system in R. rubrum is the first in which both the target protein and modifying enzymes as well as their structural genes have been isolated, making it the model system of choice for analysis of this post-translational regulatory mechanism.

Functional Characterization of Three GlnB Homologs in the Photosynthetic Bacterium Rhodospirillum rubrum: Roles in Sensing Ammonium and Energy Status

The GlnB (P(II)) protein, the product of glnB, has been characterized previously in the photosynthetic bacterium Rhodospirillum rubrum. Here we describe identification of two other P(II) homologs in this organism, GlnK and GlnJ. Although the sequences of these three homologs are very similar, the molecules have both distinct and overlapping functions in the cell. While GlnB is required for activation of NifA activity in R. rubrum, GlnK and GlnJ do not appear to be involved in this process. In contrast, either GlnB or GlnJ can serve as a critical element in regulation of the reversible ADP ribosylation of dinitrogenase reductase catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase-activating glycohydrolase (DRAG) regulatory system. Similarly, either GlnB or GlnJ is necessary for normal growth on a variety of minimal and rich media, and any of the proteins is sufficient for normal posttranslational regulation of glutamine synthetase. Surprisingly, in their regulation of the DRAT/DRAG system, GlnB and GlnJ appeared to be responsive not only to changes in nitrogen status but also to changes in energy status, revealing a new role for this family of regulators in central metabolic regulation.

Incorporation of Iron and Sulfur from NifB Cofactor into the Iron-Molybdenum Cofactor of Dinitrogenase

NifB-co is an iron- and sulfur-containing precursor to the iron-molybdenum cofactor (FeMo-co) of dinitrogenase. The synthesis of NifB-co requires at least the product of the nifB gene. Incorporation of 55Fe and 35S from NifB-co into FeMo-co was observed only when all components of the in vitro FeMo-co synthesis system were present. Incorporation of iron and sulfur from NifB-co into dinitrogenase was not observed in control experiments in which the apodinitrogenase (lacking FeMo-co) was initially activated with purified, unlabeled FeMo-co or in assays where NifB-co was oxygen-inactivated prior to addition to the synthesis system. These data clearly demonstrate that iron and sulfur from active NifB-co are specifically incorporated into FeMo-co of dinitrogenase and provide direct biochemical identification of an iron-sulfur precursor of FeMo-co. Under different in vitro FeMo-co synthesis conditions, iron and sulfur from NifB-co were associated with at least two other proteins (NIFNE and gamma) that are involved in the formation of active dinitrogenase. The results presented here suggest that multiple FeMo-co processing steps might occur on NIFNE and that FeMo-co synthesis is most likely completed prior to the association of FeMo-co with gamma.

Reversible ADP-ribosylation as a mechanism of enzyme regulation in procaryotes

Several cases of ADP-ribosylation of endogenous proteins in procaryotes have been discovered and investigated. The most thoroughly studied example is the reversible ADP-ribosylation of the dinitrogenase reductase from the photosynthetic bacteriumRhodospirillum rubrum and related bacteria. A dinitrogenase reductase ADP-ribosyltransferase (DRAT) and a dinitrogenase reductase ADP-ribose glycohydrolase (DRAG) fromR. rubrum have been isolated and characterized. The genes for these proteins have been isolated and sequences and show little similarity to the ADP-ribosylating toxins. Other targets for endogenous ADP-ribosylation by procaryotes include glutamine synthetase inR. rubrum andRhizobium meliloti and undefined proteins inStreptomyces griseus andPseudomonas maltophila.

Mutagenesis and Functional Characterization of the glnB, glnA, and nifA Genes from the Photosynthetic Bacterium Rhodospirillum rubrum

Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation of nif expression and posttranslational regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase-dinitrogenase reductase-activating glycohydrolase) system. We report here the characterization of glnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for P(II)) were constructed. While P(II)-Y51F showed a lower nitrogenase activity than that of wild type, a P(II) deletion mutant showed very little nif expression. This effect of P(II) on nif expression is apparently the result of a requirement of P(II) for NifA activation, whose activity is regulated by NH(4)(+) in R. rubrum. The modification of glutamine synthetase (GS) in these glnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of P(II) might exist in R. rubrum and regulate the modification of GS. P(II) also appears to be involved in the regulation of DRAT activity, since an altered response to NH(4)(+) was found in a mutant expressing P(II)-Y51F. The adenylylation of GS plays no significant role in nif expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH(4)(+) and darkness treatments.

Purification and Characterization of Membrane-associated CooC Protein and Its Functional Role in the Insertion of Nickel into Carbon Monoxide Dehydrogenase from Rhodospirillum rubrum

The accessory protein CooC, which contains a nucleotide-binding domain (P-loop) near the N terminus, participates in the maturation of the nickel center of carbon monoxide dehydrogenase (CODH). In this study, CooC was purified from the chromatophore membranes of Rhodospirillum rubrum with a 3,464-fold purification and a 0.8% recovery, and its biochemical properties were characterized. CooC is a homodimer with a molecular mass of 61–63 kDa, contains less than 0.1 atom of Ni2+ or Fe2+ per dimer, and has a λmax at 277.5 nm (ε277.5 32.1 mm −1cm−1) with no absorption peaks at the visible region. CooC catalyzes the hydrolysis of ATP and GTP with K m values of 24.4 and 26.0 μm andV max values of 58.7 and 3.7 nmol/min/mg protein for ATP and GTP hydrolysis, respectively. The P-loop mutated form of K13Q CooC was generated by site-specific replacement of lysine by glutamine and was purified according to the protocol for wild-type CooC purification. The K13Q CooC was inactive both in ATP hydrolysis andin vivo nickel insertion. In vitro nickel activation of apoCODH in the cell extracts from UR2 (wild type) and UR871 (K13Q CooC) showed that activation of nickel-deficient CODH was enhanced by CooC and dependent upon ATP hydrolysis. The overall results suggest that CooC couples ATP hydrolysis with nickel insertion into apoCODH. On the basis of our results and models for analogous systems, the functional roles of CooC in nickel processing into the active site of CODH are presented.