High resolution x-ray diffraction data from crystals of the Rhodobacter sphaeroides photosynthetic reaction center (RC) have been collected at cryogenic temperature in the dark and under illumination, and the structures were refined at 2.2 and 2.6 angstrom resolution, respectively. In the charge-separated D+QAQB- state (where D is the primary electron donor (a bacteriochlorophyll dimer), and QA and QB are the primary and secondary quinone acceptors, respectively), QB- is located approximately 5 angstroms from the QB position in the charge-neutral (DQAQB) state, and has undergone a 180 degrees propeller twist around the isoprene chain. A model based on the difference between the two structures is proposed to explain the observed kinetics of electron transfer from QA-QB to QAQB- and the relative binding affinities of the different ubiquinone species in the QB pocket. In addition, several water channels (putative proton pathways) leading from the QB pocket to the surface of the RC were delineated, one of which leads directly to the membrane surface.

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...