Redox-Dependent Structural Changes in the Nitrogenase P-Cluster^,

Abstract

The effect of nitrogenase 'switch-off effecters on the concentration of NAD(P)H in ModospirilZum rubrum G-9 was investigated by fluorescence. A rapid decrease in fluorescence was observed when cells, either N,-grown or nitrogen-starved, were subjected to the effecters, but not when sodium chloride or Tris buffer was added. No effects on the fluorescence were observed in non-nitrogen fixing cultures except when NAD+ was added. The results strongly indicate that the redox state of the pyridine nucleotide pool affects the control of the regulation of nitrogenase activity in R rubrum. Nitrogen fixation is carried out by a number of bacteria, in a reaction catalyzed by nitrogenase, which consists of two proteins , dinitrogenase and dinitrogenase reductase. Electrons are transferred from dinitrogenase reductase to dinitrogenase in a reaction requiring hydrolysis of MgATP [l]. In a number of phototrophs and some species of Azospirillum, nitrogen tixa-tion is regulated not only genetically but also metabolically [2,3]. In the photosynthetic bacterium R rubrum, nitrogenase activity is regulated by reversible inhibition, a phenomenon referred to as the 'switch-off effect [4]. At the molecular level this effect is due to reversible modification of dinitrogenase reductase by ADP-ribosylation of one of its two identical sub-units on an arginine residue, Arg-101, when the cells are subjected to darkness, ammonium ions, glutamine, asparagine or oxygen [2,3]. Other switch-off effecters are carbonyl cyanide m-chlorophenylhydrazone (CCCP) and phenazine metho-sulphate (PMS) [5]. The modification of dinitrogenase reduc-tase is catalyzed by dinitrogenase reductase ADP-ribosyl trans-ferase (DRAT) with NAD' as the donor of ADP-ribose [2]. The reverse reaction is catalyzed by dinitrogenase reductase activating glycohydrolase (DRAG) [2], which requires ATP and a divalent cation such as manganese or ferrous iron [6]. The internal signal between the switch-off effector and DRAGlDRAT has not yet been identified, but the nitrogen status and the NAD(P)+/NAD(P)H ratio have been suggested to be involved in the regulation of these enzymes. We have previously shown that adding NAD+ to a nitrogen-fixing culture of R rubrum results in a reversible decrease in activity, an effect dependent on light intensity; at lower light intensities the effect is more pronounced [7l. The effect of NAD' can also be seen in nitrogen-starved cells which cannot be 'switched off by any of the other effecters tested. We have previously suggested that an increase in the NAD' concentration could be involved in the control of the activities of DRAG and especially DRAT, and that the nitrogen status of the cell is also of possible *Corresponding author. Fax: (46) 8 15 77 94. importance [7l. An increase in the NAD' concentration could also act as a direct signal for DRAT activity since the enzyme is NADC dependent, having a high & for NAD' with dinitro-genase reductase from R rubrum [2,7]. In this investigation we have studied the influence of switch-off effecters on the NA...

FeMo cofactor maturation on NifEN

“…The homodimeric Fe protein has one nucleotide-binding site per subunit and a single [4Fe-4S] cluster bridged between the two subunits. The ␣ 2 ␤ 2 -tetrameric MoFe protein contains two unique metal clusters per ␣␤-subunit: the [8Fe-7S] P-cluster (9), which is located at the ␣␤-interface and ligated to six protein residues; and the [Mo-7Fe-9S-X-homocitrate] (the identity of X is unknown but is considered to be C, O, or N; ref. 10) FeMo cofactor (FeMoco), which is situated within the ␣-subunit and bound to only two protein residues and an exogenous homocitrate ligand.…”

“…10) FeMo cofactor (FeMoco), which is situated within the ␣-subunit and bound to only two protein residues and an exogenous homocitrate ligand. Both P-cluster and FeMoco are composed of smaller substructures: the P-cluster comprises two [4Fe-4S] subclusters that share a 6 -sulfide (9) and FeMoco consists of [Mo-3Fe-3S] and [4Fe-3S] subcubanes that are bridged by three 2 -sulfides and share a central 6 -light atom (10). These metal clusters are essential for nitrogenase reaction, a process that involves ATP-dependent electron transfer from the [4Fe-4S] cluster of the Fe protein to the P-cluster of the MoFe protein and finally to FeMoco where substrate reduction takes place, and consequently become the major subjects in the vigorous studies of nitrogenase catalysis (1)(2)(3)(4)(5)(6)(7)(8).…”

P-cluster maturation on nitrogenase MoFe protein

“…The homodimeric Fe protein has one ATP binding site per subunit and a single [4Fe-4S] cluster bridged in between the subunits, whereas the ␣ 2 ␤ 2 -tetrameric MoFe protein (Fig. 1A) contains two unique clusters per ␣␤-subunit pair: the [8Fe-7S] P-cluster (6), which is located at the ␣␤-subunit interface, and the [Mo-7Fe-9S-X-homocitrate] † iron-molybdenum cofactor (FeMoco) (7), which is positioned within the ␣-subunit. Nitrogenase catalysis involves a series of complex formation and dissociation between the Fe protein and the MoFe protein, during which process the electrons are sequentially transferred from the [4Fe-4S] cluster of the Fe protein, through the P-cluster, to the FeMoco within the MoFe protein, where substrate reduction eventually occurs.…”

“…Catalytically, it serves as a ''hub'' that mediates the shuffling of electrons between the metal centers of the Fe protein and the MoFe protein. Structurally, it represents a high-nuclearity, Fe/ S-only cluster that can be viewed as two [4Fe-4S] subclusters sharing a 6 -sulfide. Such a ''modular'' composition suggests that the P-cluster is formed through the fusion of its substructural units, a reaction mechanism that is well established in synthetic inorganic chemistry (8) and further supported by recent advances toward successful synthesis of P-cluster topologs (9)(10)(11)(12).…”

Nitrogenase reactivity with P-cluster variants

“…f Although encoded by different structural genes, all three nitrogenases are comprised of two essential component metalloproteins, component 1 (MoFe, VFe, or FeFe protein) and component 2 (Fe protein). g The homodimeric Fe protein of the Mo-nitrogenase has one [4Fe-4S] cluster bridged between the two subunits, whereas the ␣ 2 ␤ 2 -tetrameric MoFe protein contains two unique metal clusters per ␣␤-subunit: the [8Fe-7S] P-cluster (14), which is located at the ␣␤-interface, and the [Mo-7Fe-9S-X-homocitrate] h FeMo cofactor (FeMoco), which is situated within the ␣-subunit. ATP-dependent electron transfer is believed to proceed from the [4Fe-4S] cluster of the Fe protein to the P-cluster of the MoFe protein and finally to FeMoco where substrate reduction takes place.…”