Iron-sulfur [Fe-S] clusters are ubiquitous and evolutionary ancient prosthetic groups that are required to sustain fundamental life processes. Owing to their remarkable structural plasticity and versatile chemical/electronic features [Fe-S] clusters participate in electron transfer, substrate binding/activation, iron/sulfur storage, regulation of gene expression, and enzyme activity. Formation of intracellular [Fe-S] clusters does not occur spontaneously but requires a complex biosynthetic machinery. Three different types of [Fe-S] cluster biosynthetic systems have been discovered, and all of them are mechanistically unified by the requirement for a cysteine desulfurase and the participation of an [Fe-S] cluster scaffolding protein. Important mechanistic questions related to [Fe-S] cluster biosynthesis involve the molecular details of how [Fe-S] clusters are assembled on scaffold proteins, how [Fe-S] clusters are transferred from scaffolds to target proteins, how various accessory proteins participate in [Fe-S] protein maturation, and how the biosynthetic process is regulated.

Iron-sulfur cluster biosynthesis in both prokaryotic and eukaryotic cells is known to be mediated by two highly conserved proteins, termed IscS and IscU in prokaryotes. The homodimeric IscS protein has been shown to be a cysteine desulfurase that catalyzes the reductive conversion of cysteine to alanine and sulfide. In this work, the time course of IscS-mediated Fe-S cluster assembly in IscU was monitored via anaerobic anion exchange chromatography. The nature and properties of the clusters assembled in discrete fractions were assessed via analytical studies together with absorption, resonance Raman, and Mössbauer investigations. The results show sequential cluster assembly with the initial IscU product containing one [2Fe-2S](2+) cluster per dimer converting first to a form containing two [2Fe-2S](2+) clusters per dimer and finally to a form that contains one [4Fe-4S](2+) cluster per dimer. Both the [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU are reductively labile and are degraded within minutes upon being exposed to air. On the basis of sequence considerations and spectroscopic studies, the [2Fe-2S](2+) clusters in IscU are shown to have incomplete cysteinyl ligation. In addition, the resonance Raman spectrum of the [4Fe-4S](2+) cluster in IscU is best interpreted in terms of noncysteinyl ligation at a unique Fe site. The ability to assemble both [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU supports the proposal that this ubiquitous protein provides a scaffold for IscS-mediated assembly of clusters that are subsequently used for maturation of apo Fe-S proteins.

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...