Occurrence, classification, and biological function of hydrogenases: an overview.

Energy conservation via electron bifurcating ferredoxin reduction and proton/Na+ translocating ferredoxin oxidation

Production of hydrogen by anaerobes, facultative anaerobes, aerobes, methylotrophs, and photosynthetic bacteria is possible. Anaerobic Clostridia are potential producers and immobilized C. butyricum produces 2 mol H2/mol glucose at 50% efficiency. Spontaneous production of H2 from formate and glucose by immobilized Escherichia coli showed 100% and 60% efficiencies, respectively. Enterobactericiae produces H2 at similar efficiency from different monosaccharides during growth. Among methylotrophs, methanogenes, rumen bacteria, and thermophilic archae, Ruminococcus albus, is promising (2.37 mol/mol glucose). Immobilized aerobic Bacillus licheniformis optimally produces 0.7 mol H2/mol glucose. Photosynthetic Rhodospirillum rubrum produces 4, 7, and 6 mol of H2 from acetate, succinate, and malate, respectively. Excellent productivity (6.2 mol H2/mol glucose) by co-cultures of Cellulomonas with a hydrogenase uptake (Hup) mutant of R. capsulata on cellulose was found. Cyanobacteria, viz., Anabaena, Synechococcus, and Oscillatoria sp., have been studied for photoproduction of H2. Immobilized A. cylindrica produces H2 (20 ml/g dry wt/h) continually for 1 year. Increased H2 productivity was found for Hup mutant of A. variabilis. Synechococcus sp. has a high potential for H2 production in fermentors and outdoor cultures. Simultaneous productions of oxychemicals and H2 by Klebseilla sp. and by enzymatic methods were also attempted. The fate of H2 biotechnology is presumed to be dictated by the stock of fossil fuel and state of pollution in future.

Microbial production of hydrogen : an overview

Clostridium kluyveri is unique among the clostridia; it grows anaerobically on ethanol and acetate as sole energy sources. Fermentation products are butyrate, caproate, and H2. We report here the genome sequence of C. kluyveri, which revealed new insights into the metabolic capabilities of this well studied organism. A membrane-bound energy-converting NADH:ferredoxin oxidoreductase (RnfCDGEAB) and a cytoplasmic butyryl-CoA dehydrogenase complex (Bcd/EtfAB) coupling the reduction of crotonyl-CoA to butyryl-CoA with the reduction of ferredoxin represent a new energy-conserving module in anaerobes. The genes for NAD-dependent ethanol dehydrogenase and NAD(P)-dependent acetaldehyde dehydrogenase are located next to genes for microcompartment proteins, suggesting that the two enzymes, which are isolated together in a macromolecular complex, form a carboxysome-like structure. Unique for a strict anaerobe, C. kluyveri harbors three sets of genes predicted to encode for polyketide/nonribosomal peptide synthetase hybrides and one set for a nonribosomal peptide synthetase. The latter is predicted to catalyze the synthesis of a new siderophore, which is formed under iron-deficient growth conditions.

https://www.pnas.org/content/pnas/105/6/2128.full.pdf

The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features

The fixation of inorganic carbon into organic material (autotrophy) is a prerequisite for life and sets the starting point of biological evolution. In the extant biosphere the reductive pentose phosphate (Calvin-Benson) cycle is the predominant mechanism by which many prokaryotes and all plants fix CO2 into biomass. However, the fact that five alternative autotrophic pathways exist in prokaryotes is often neglected. This bias may lead to serious misjudgments in models of the global carbon cycle, in hypotheses on the evolution of metabolism, and in interpretations of geological records. Here, I review these alternative pathways that differ fundamentally from the Calvin-Benson cycle. Revealingly, these five alternative pathways pivot on acetyl-coenzyme A, the turntable of metabolism, demanding a gluconeogenic pathway starting from acetyl-coenzyme A and CO2. It appears that the formation of an activated acetic acid from inorganic carbon represents the initial step toward metabolism. Consequently, biosynthese...

Alternative Pathways of Carbon Dioxide Fixation: Insights into the Early Evolution of Life?

DNA sequence analysis of a 12236 bp fragment, which is located upstream of nifE in Rhodobacter capsulatus nif region A, revealed the presence of ten open reading frames With the exception of fdxC and fdxN, which encode a plant-type and a bacterial-type ferredoxin, the deduced products of these coding regions exhibited no significant homology to known proteins Analysis of defined insertion and deletion mutants demonstrated that six of these genes were required for nitrogen fixation Therefore, we propose to call these genes rnfA, rnfB, rnfC, rnfD, rnfE and rnfF (for Rhodobacter nitrogen fixation) Secondary structure predictions suggested that the rnf genes encode four potential membrane proteins and two putative iron-sulphur proteins, which contain cysteine motifs (C-X2-C-X2-C-X3-C-P) typical for [4Fe--4S] proteins Comparison of the in vivo and in vitro nitrogenase activities of fdxN and rnf mutants suggested that the products encoded by these genes are involved in electron transport to nitrogenase In addition, these mutants were shown to contain significantly reduced amounts of nitrogenase The hypothesis that this new class of nitrogen fixation genes encodes components of an electron transfer system to nitrogenase was corroborated by analysing the effect of metronidazole Both the fdxN and rnf mutants had higher growth yields in the presence of metronidazole than the wild type, suggesting that these mutants contained lower amounts of reduced ferredoxins

Identification of a new class of nitrogen fixation genes in Rhodobacter capsulatus: a putative membrane complex involved in electron transport to nitrogenase

In Rhodobacter capsulatus, the hupL gene encoding the large subunit of the uptake-hydrogenase (Hup) enzyme complex was mutated by insertion of an interposon. The mutant neither synthesized an active hydrogenase nor grew photoautotrophically. Under conditions of nitrogen (N) limitation, photoheterotrophic cultures of the wild type and the mutant evolved H2 by activity of the nitrogenase enzyme complex. When grown with glutamate as an N source and either D,L-malate or L-lactate as carbon sources, the efficiency of H2 production by the HupL mutant was higher than 90%, whereas wild-type cultures exhibited efficiencies of 54% (with D,L-malate) and 64% (with L-lactate), respectively. With NH4+ as the N source, efficiencies of H2 production were 70% (mutant) and 52% (wild type).

Optimizing photoheterotrophic H2 production by Rhodobacter capsulatus upon interposon mutagenesis in the hupL gene

The production of biomass, polysaccharide storage material and H2 from malate was studied in the wild-type and mutants RdcI, RdcII and RdcI/cII of Rhodobacter capsulatus. The mutants are defective in either copy I, copy II or both copies of the nitrogenase genes nifA and nifB. Stationary phase levels of biomass, polysaccharide and H2 were determined in phototrophic batch cultures grown with 30 mM of d,l-malate and either 2, 5, or 8 mM of ammonium or 7 mM of glutamate. Calculation of the amounts of malate converted into the three products revealed that, at 8 mM of ammonium and 7 mM of glutamate, malate consumption and product formation were balanced. But with decreasing ammonium concentrations malate not converted into biomass was utilized with decreasing efficiency in polysaccharide and H2 formation. This suggests formation of unknown products at the lower ammonium concentrations. Under conditions of optimal N supply, 80% of the malate not used for biomass production was converted by the wild-type and strain RdcII to H2 and CO2. Mutant RdcI exhibited slightly decreased H2 production. The double mutant did not evolve H2 but accumulated increased amounts of polysaccharide. However, the amounts of polysaccharide were lower than should be expected if all of the spare malate, not utilized by the double mutant for H2 production, was converted into storage material. This and incomplete conversion of malate into known products at low ammonium supplies suggests that polysaccharide accumulation does not compete with the process of H2 formation for malate.

The relationship of biomass, polysaccharide and H2 formation in the wild-type and nifA/nifB mutants of Rhodobacter capsulatus

Abstract In chemostat cultures of Rhodobacter capsulatus, growing aerobically in darkness, in situ nitrogen fixation occurred at significantly lower oxygen concentrations than the acetylene reduction activity of nitrogenase, as determined with samples from the culture under opti mum assay conditions. In contrast to nitrogenase of cultures growing diazotrophically at low oxygen, nitrogenase present in inactive form at the higher oxygen concentrations could not be activated to fix nitrogen by increasing i.) the energy supply by illuminating the cultures, ii.) the supply of cells with electron donor and iii.) cellular respiration. These results suggest that oxygen controls cellular nitrogen fixation directly rather than indirectly by interfering with the general metabolism. On this basis, the sensitivity toward oxygen of nif gene expression as well as of nitrogenase polypeptide accumulation was studied. The nifH promoter was active up to about 40% air saturation, exhibiting a biphasic sensitivity to oxygen, i.e. steep decreases in activity between 1 to 2% and between 10 to about 20% air saturation. A similar behaviour was observed with respect to cellular levels of both polypeptides of component 1 (Rc1) of nitrogenase, which were accumulated up to about 20% air saturation. Component 2 (Rc2) was less oxygen-sensitive than Rc1, its steady state level reached a maximum at 2% air saturation. A sudden increase in the ambient oxygen concentration, i. e. oxygen stress, revealed that, in vivo, nitrogenase was fairly stable against oxygen damage. Modification of Rc2 by ADP-ribosylation occurred under ammonium shock conditions, regardless of whether the cells fixed nitrogen or not. But it was not observed with cultures growing aerobically in the dark at steady state or under oxygen stress. Results obtained with mutant W107I of R. capsulatus lacking the modifying/demodifying enzymes supported the conclusion that modifi cation of Rc2 was not required for the inactivation of nitrogenase by oxygen.

The Oxygen Sensitivity of Nitrogenase in Rhodobacter capsulatus: Repression and Inactivation

Rhodobacter capsulatus was grown chemotrophically in the dark in oxygen-regulated chemostat culture and in the presence of limiting amounts of fixed N. When the oxygen partial pressure was varied, in situ nitrogen fixation occurred only at 1% of air saturation of the medium. By contrast, nitrogenase proteins and their activity measured in the absence of oxygen could be detected up to 30% of air saturation. This revealed that expression of nitrogenase is much less sensitive toward oxygen than the in situ function of the enzyme. At oxygen partial pressures > 1% of air saturation, the degree of modification of the Fe protein of nitrogenase was increased. Light was of no stimulatory effect on both the activity and the expression of nitrogenase. This holds true for growth at 1% or 5% of air saturation. At 5% of air saturation, however, high illumination enhanced the inhibitory effect of oxygen on nitrogenase formation.

Andreas Jahn

Papers

Identification of a new class of nitrogen fixation genes in Rhodobacter capsulatus: a putative membrane complex involved in electron transport to nitrogenase

Optimizing photoheterotrophic H2 production by Rhodobacter capsulatus upon interposon mutagenesis in the hupL gene

The relationship of biomass, polysaccharide and H2 formation in the wild-type and nifA/nifB mutants of Rhodobacter capsulatus

The Oxygen Sensitivity of Nitrogenase in Rhodobacter capsulatus: Repression and Inactivation

Activity and expression of nitrogenase in Rhodobacter capsulatus under aerobiosis in the dark and in the light