A three-dimensional structure for the monomeric iron-containing hydrogenase (CpI) from Clostridium pasteurianum was determined to 1.8 angstrom resolution by x-ray crystallography using multiwavelength anomalous dispersion (MAD) phasing. CpI, an enzyme that catalyzes the two-electron reduction of two protons to yield dihydrogen, was found to contain 20 gram atoms of iron per mole of protein, arranged into five distinct [Fe-S] clusters. The probable active-site cluster, previously termed the H-cluster, was found to be an unexpected arrangement of six iron atoms existing as a [4Fe-4S] cubane subcluster covalently bridged by a cysteinate thiol to a [2Fe] subcluster. The iron atoms of the [2Fe] subcluster both exist with an octahedral coordination geometry and are bridged to each other by three non-protein atoms, assigned as two sulfide atoms and one carbonyl or cyanide molecule. This structure provides insights into the mechanism of biological hydrogen activation and has broader implications for [Fe-S] cluster structure and function in biological systems.

The splitting of dinitrogen (N2) and reduction to ammonia (NH3) is a kinetically complex and energetically challenging multistep reaction. In the Haber-Bosch process, N2 reduction is accomplished at high temperature and pressure, whereas N2 fixation by the enzyme nitrogenase occurs under ambient conditions using chemical energy from adenosine 5'-triphosphate (ATP) hydrolysis. We show that cadmium sulfide (CdS) nanocrystals can be used to photosensitize the nitrogenase molybdenum-iron (MoFe) protein, where light harvesting replaces ATP hydrolysis to drive the enzymatic reduction of N2 into NH3 The turnover rate was 75 per minute, 63% of the ATP-coupled reaction rate for the nitrogenase complex under optimal conditions. Inhibitors of nitrogenase (i.e., acetylene, carbon monoxide, and dihydrogen) suppressed N2 reduction. The CdS:MoFe protein biohybrids provide a photochemical model for achieving light-driven N2 reduction to NH3.

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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...