M. Geoffrey Yates

Nitrogen Fixation and Hydrogen Metabolism in Cyanobacteria

SUMMARY This review summarizes recent aspects of (di)nitrogen fixation and (di)hydrogen metabolism, with emphasis on cyanobacteria. These organisms possess several types of the enzyme complexes catalyzing N2 fixation and/or H2 formation or oxidation, namely, two Mo nitrogenases, a V nitrogenase, and two hydrogenases. The two cyanobacterial Ni hydrogenases are differentiated as either uptake or bidirectional hydrogenases. The different forms of both the nitrogenases and hydrogenases are encoded by different sets of genes, and their organization on the chromosome can vary from one cyanobacterium to another. Factors regulating the expression of these genes are emerging from recent studies. New ideas on the potential physiological and ecological roles of nitrogenases and hydrogenases are presented. There is a renewed interest in exploiting cyanobacteria in solar energy conversion programs to generate H2 as a source of combustible energy. To enhance the rates of H2 production, the emphasis perhaps needs not to be on more efficient hydrogenases and nitrogenases or on the transfer of foreign enzymes into cyanobacteria. A likely better strategy is to exploit the use of radiant solar energy by the photosynthetic electron transport system to enhance the rates of H2 formation and so improve the chances of utilizing cyanobacteria as a source for the generation of clean energy.

The hydrogen cycle in nitrogen-fixing Azotobacter chroococcum.

H2 will support nitrogenase activity (C2H2 reduction) in Azotobacter chroococcum with or without added carbon substrate. Results show that H2 is metabolised to transfer electrons to nitrogenase and to the respiratory chain to produce ATP. H2-supported nitrogenase activity is most significant at low carbon substrate concentrations, but also occurs at saturating concentration. Continuous cultures of N2-fixing A. chroococcum evolved H2 from nitrogenase under O2-N2- and C-limited conditions. This H2 represented a significant proportion of nitrogenase activity. Hydrogenase activity was consistently high under C-limited conditions, but low or undetectable under O2- and N2-limitations. Pre-treatment with 40 per cent C2H2 inhibited hydrogenase activity in C-limited cultures, and H2 evolution increased under air and under Ar:O2 (4:1) mixtures. We deduce that hydrogenase : I, recycles H2 produced by nitrogenase to provide electrons and energy for N2 reduction: II, supports respiratory protection for nitrogenase under C-limited conditions, and III, does not act to prevent any inhibition of N2 reduction by H2 produced by nitrogenase. A scheme for the H2 cycle in N2-fixing A. chroococcum is proposed.

Nitrogenases of Klebsiella pneumoniae and Azotobacter chroococcum: Complex formation between the component proteins

1. Sedimentation velocity analyses of mixtures of highly purified component proteins of Azotobacter chroococcum are consistent with the formation of a tight 1 : 1 complex in the absence of Na2 S2 O4. 1 : 1 complex formation between complementary proteins from A. chroococcum and Klebsiella pneumoniae was also observed. The addition of 5 mM Na2 S2 O4 weakened the interaction between the A. chroococcum proteins and also the interaction between complementary proteins of A. chroococcum and K. pneumoniae. 2. Steady-state kinetic data for acetylene reduction at low protein concentrations have been used to calculate association constants at 30 degrees C for the 1 : 1 protein complexes of nitrogenase proteins from A. chroococcum, K. pneumoniae and mixtures of complementary proteins from both organisms. Values centered around 3 - 10(7) M-1 were obtained. 3. The temperature dependence of the association constant for the complex formed by the K. pneumoniae proteins exhibited a sharp break at 17 degrees C with deltaH = 0 and deltaH = 418 kJ - mol-1 above and below 17 degrees C, respectively. 4. The Arrhenius plot for acetylene reduction by the complex formed by the K. pneumoniae proteins was linear over the range 12-40 degrees C with deltaH = 80 kJ - mol-1.

Direct electrochemistry in the characterisation of redox proteins: Novel properties of Azotobacter 7Fe ferredoxin

Fast diffusion‐dominated electron transfer between Azotobacter chroococcum 7Fe ferredoxin, FdI, and pyrolytic graphite ‘edge’ electrodes, promoted by aminoglycosides, permits detailed voltammetric studies and preparation of oxidation states inaccessible by chemical titration. The [3Fe‐4S] cluster exhibits pH dependent E values (30°C); E (alkaline) = 460 ± 10 mV vs NHE, −dE /d(pH) = 55 mV, pK = 7.8. The [4Fe‐4S] cluster is characterised by an unusually low reduction potential, −645 ± 10 mV vs NHE, at pH 8.3, with a slight pH dependence, −dE /d(pH) ∼25 mV over the pH range 8.5‐7.0. No redox couple is observed at potentials between −300 and +600 mV vs NHE. This shows that the [4Fe‐4S] cluster is not an HIPIP‐type centre. The electron paramagnetic resonance spectrum, centred at g = 1.93, of the product resulting from bulk electrolysis at −835 mV is assigned to a [4Fe‐4S]+ cluster interacting magnetically with a reduced [3Fe‐4S] cluster.

Purification of the activating enzyme for the Fe-protein of nitrogenase from Azospirillum brasilense

Abstract The activating enzyme for the Fe-protein of nitrogenase from Azospirillum brasilense has been purified to near homogeneity. The procedure includes ion-exchange chromatography, chromatofocusing and gel filtration. The M r of the purified enzyme was determined to be 33 500 on SDS-polyacrylamide gel electrophoresis. The purified enzyme was compared with the acticating enzyme from Rhodospirillum rubrum .

Nitrogenases and Hydrogenases in Cyanobacteria

Apart from the conventional, Mo-containing nitrogenase, the cyanobacterium Anabaena variabilis can express at least two alternative N2-fixing enzyme complexes. This cyanobacterium grows with V in a Mo-deficient medium. The nitrogenase then expressed reduces C2H2 partly beyond C2H4 to C2H6 and produces more H2 than the Mo-enzyme (Kentemich et al., 1988; Yakunin et al., 1991). Genes for this V-nitrogenase, vnfDGK (Thiel, 1993) and nifB (commonly used for both nitrogenases) (Lyons and Thiel, 1995) have subsequently been cloned, mapped and sequenced. It is generally agreed that A. variabilis contains a V-nitrogenase. In addition, there is physiological evidence that A. variabilis also contains an Fe-only nitrogenase (Kentemich et al., 1991). After an extensive state of N-deprivation where the cells totally bleached due to the utilization of phycobilins as N-reserve, A. variabilis restarted to grow slowly. The cultures reduced C2H2 partly to C2H6 and produced H2 with a high rate. Determination by atomic absorption spectrometry showed that the concentration of Mo in the medium was <10 nM and of V <20 nM and likely too low for the expression of a Mo- or a V-nitrogenase. The cells showed an outburst in C2H6- and H2 -formations 3 h after the addition of Mo to the medium which typically happens with the Fe-only hydrogenase from Azotobacter vinelandii (Pau et al., 1989). Hybridizations of the anfH and nifH probes with genomic DNA from A. variabilis gave at least two distinct bands (Kentemich et al., 1991). Supporting evidence for the existence of an Fe-only nitrogenase came from Ni et al., (1990). The gene set coding for this enzyme complex has, however, not yet been found which leaves, of course, doubts about the meanings of these physiological results.