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...

Recombinant human ferrochelatase has been expressed in Escherichia coli and purified to homogeneity. Metal analyses revealed approximately 2 mol of non-heme Fe per mol of the purified enzyme (M(r) = 40,000). The UV-visible absorption spectrum of the purified enzyme consists of a protein absorption at 278 nm (epsilon approximately 90,000 M-1 cm-1) and bands at 330 nm (epsilon approximately 24,000 M-1 cm-1), 460 nm (shoulder, epsilon approximately 11,000 M-1 cm-1), and 550 nm (shoulder, epsilon approximately 9000 M-1 cm-1) that are indicative of a [2Fe-2S]2+ cluster. The spectra show an additional band at 415 nm that varied in intensity for different preparations and is attributed, at least in part, to a minor component of enzyme-associated high-spin Fe(III) heme. The presence of a single [2Fe-2S]2+,+ cluster as a redox active component of human ferrochelatase was confirmed by variable-temperature MCD and EPR studies of the dithionite-reduced enzyme which showed the presence of a S = 1/2 [2Fe-2S]+ cluster in addition to residual high spin Fe(II) heme. The reduced enzyme exhibits a S = 1/2 EPR signal, g = 2.00, 1.94, 1.91 accounting for 0.75 +/- 0.25 spins/molecule, that readily saturates at low microwave powers below 10 K but is observable without significant broadening at temperatures up to 100 K. The Fe-S cluster is labile and gradually disappears over period of 24 h, with concomitant loss of enzyme activity, when the enzyme is stored aerobically at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)