1. Several low-potential electron carriers from different sources can be photoreduced by the system 3,l O-dimethyl-5-deazaisoalloxazine/N-tris(hydroxymethyl)methylglycine. The carriers studied were flavodoxin, ferredoxin 1 and iron-sulfur protein I1 from Azotohacter vinelundii and the flavodoxins from Desulfovibrio vulgaris and Peptostreptococcus elsdenii.2. Electron transport to A . vinelandii nitrogenase was studied, employing different preparations of the enzyme: a crude complex; a complex reconstituted from the 0.27 M and 0.38 M NaCl fractions after DEAE-cellulose chromatography; a complex reconstituted from the 0.27 M and 0.38 M NaCl fraction plus iron-sulfur protein I1 purified from the 0.15 M NaCl fraction. Of all photoreduced carriers tested, only flavodoxin hydroquinone from A . vinelundii catalyzes significant electron transport to these complexes.3. The time course of oxidation of substrate-amounts of A . vinelandii flavodoxin hydroquinone by catalytic amounts of crude nitrogenase complex shows three characteristic phases : an initial lag phase (l), a phase with constant rate over a range of redox potentials (2) and a final phase with rapidly declining rate (3). It was shown that the Fe-S protein I1 is responsible for the lag phase; the potential where phase 2 changes into phase 3 is at a higher value in the presence of Fe-S protein 11. Pre-reduction of the enzyme photochemically abolishes phase 1 and causes phase 2 to proceed at a higher rate. The rate in phase 2 can be enhanced also by lowering the 'starting potential' of the flavodoxin hydroquinone/semiquinone couple. 4. A . vinelandii flavodoxin shuttles between its hydroquinone and semiquinone forms during steady-state electron transfer. Over 90 % of the reducing equivalents is recovered in ethylene formed from acetylene. The concentrations of flavodoxin hydroquinone to give half-maximum rate in the acetylene reduction assay is 3 -6 pM. A scheme is proposed for the regulation of electron donation to nitrogenase.It is well established that pyruvate is a main source of reducing equivalents for nitrogenase in many aerobic nitrogen-fixing bacteria, and that electrons are usually transferred from pyruvate dehydrogenase via ferredoxin [l]. Benemann et al. [2] as well as Yoch and Arnon [3] considered ferredoxin a possible candidate as electron donor for nitrogen fixation in Azotohacter, but attempts to demonstrate pyruvate-ferredoxin oxidoreductase activity in extracts have given equivocal results [4].The role of flavodoxin in aerobic nitrogen-fixing organisms is also not certain. As in aerobic organisms it might substitute for ferredoxin under conditions of iron-deficiency. Benemann et al. [2,5] were the first to postulate a role of flavodoxin in nitrogen fixation of Azotobacter. They showed that flavodoxin could couple the reducing power of chloroplast photosystem I to nitrogenase in a cell-free extract. However, the activity obtained amounted to only a fraction of the activity with dithionite as electron donor. In addition, the specificity of the re...

Conditions are defined in which the oxygen-labile nitrogenase components from Azotohucter vinelandii can be protected against oxygen inactivation by the so-called Fe/S protein 11. It is demonstrated that oxygen protection can be achieved by complex formation of the three proteins. Complex formation was studied by gel chromatography. Only when the three proteins are in the oxidized state and MgC1, is present, can an oxygen-tolerant complex be isolated. Quantitative SDS/polyacrylamide gel electrophoresis of such complexes, yielded an average ratio of nitrogenase component 2/nitrogenase component 1 (Av2/Av,) of 2.4+ 0. Analysis in the ultracentrifuge showed that the major fraction of the reconstituted complex has a sedimentation coefficient centered around 34 S. A small fraction (< 30 7") sediments with values centered around 11 1 S. This suggests an average mass for the oxygen-stable nitrogenase complex of 1.5 MDa. Taking into account the determined stoichiometry of the individual proteins, the molecular composition of the oxygen-stable nitrogenase complex is presumably 4 molecules of Av,, 8-12 molecules of Av, and 4-6 molecules of Fe/S protein I1 containing two [2Fe-2S] clusters per dimer of 26 kDa.Introductions to scientific papers on studies on the nitrogenase proteins, often start with the statement that nitrogenase can be separated into two 02-sensitive redox proteins, or words of that kind. It is therefore noteworthy that actually so littlc work has been devoted towards a study of the 02-sensi-

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Adenosine triphosphatase activity of tobacco leaf chloroplasts in the dark was measured, using leaves of different age as determined by the position of the leaves along the stem. The activity of the chloroplast preparations strongly decreased with age, regardless of the addition of Mg2+ or Ca2+. Opposite effects of Mg2+ and Ca2+ on the activity of the chloroplasts were noted in experiments where different ratios of Mg2+/Ca2+ were applied. They were related to the age of the leaves, Ca2+ strongly stimulated the activity of the preparations from old leaves but was practically without effect in young, just expanded leaves. Mg2+ slightly stimulated the activity from old leaves while it invariably inhibited the hydrolytic activity of preparations from young leaves.

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