David J. Richardson

Introduction to the biochemistry and molecular biology of denitrification.

Publisher Summary This chapter provides an overview of the biochemistry and genetics of denitrification in such organisms. It considers the aspects of denitrification that occur in archaea and certain fungi. Denitrification has been mostly studied in Paracoccus denitrificans and Pseudomonas stutzeri and so it describes denitrification for each of these organisms in turn before considering to what extent general principles can be discerned. In recent years, high-resolution crystal structures have become available for these enzymes with the exception of the structure for NO-reductase. In general, the proteins required for denitrification are only produced under (close to) anaerobic conditions, and if anaerobically grown, cells are exposed to O2 and then the activities of the proteins are inhibited. Specialized denitrifiers, such as P. denitrificans and the denitrifying Pseudomonads, contain more than 40 genes, which encode the proteins that make up a full denitrification pathway. They include the structural genes for the enzymes and e− donors, their regulators as well as many accessory genes required for assembly, cofactor synthesis, and insertion into the enzymes. In contrast, some denitrifiers can only carry out the two central reactions of the pathway and use these activities to support growth, but the cost of maintaining this capability is a very small amount of genome space. It provides insights into the regulation of gene expression and the way in which some denitrification enzymes play different roles in bacteria.

The Role of Auxiliary Oxidants in Maintaining Redox Balance During Phototrophic Growth of Rhodobacter-Capsulatus On Propionate or Butyrate

Phototrophic growth of Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata) under anaerobic conditions with either butyrate or propionate as carbonsource was dependent on the presence of either CO2 or an auxiliary oxidant. NO-3, N2O, trimethylamine-N-oxide (TMAO) or dimethylsulphoxide (DMSO) were effective provided the appropriate anaerobic respiratory pathway was present. NO-3was reduced extensively to NO-3, TMAO to trimethylamine and DMSO to dimethylsulphide under these conditions. Analysis of culture fluids by nuclear magnetic resonance showed that two moles of TMAO or DMSO were reduced per mole of butyrate utilized and one mole of either oxidant was reduced per mole of propionate consumed. The growth rate of Rb. capsulatus on succinate or malate as carbon source was enhanced by TMAO in cultures at low light intensity but not at high light intensities. A new function for anaerobic respiration during photosynthesis is proposed: it permits reducing equivalents from reduced substrates to pass to auxiliary oxidants present in the medium. The use of CO2 or auxiliary oxidants under phototrophic conditions may be influence by the availability of energy from light. It is suggested that the nuclear magnetic resonance methodology developed could have further applications in studies of bacterial physiology.

Identification of cytochromes involved in electron transport to trimethylamine N-oxide/dimethylsulphoxide reductase in Rhodobacter capsulatus

The role of cytochromes in the electron-transport pathway to trimethylamine N -oxide (TMAO)/dimethylsulphoxide (DMSO) reductase in the photosynthetic bacterium Rhodobacter capsulatus was investigated. Reduced-minus-oxidized difference spectra in intact cells with TMAO or DMSO as oxidant revealed cytochrome absorbance changes with a maximum at 559 nm and a shoulder between 548 nm and 556 nm. The former change indicates a role for a 6-type cytochrome and the latter for a c-type cytochrome, both of which are distinct from the cytochrome bc 1 complex. Cytochrome c -556 was identified in a bacterial periplasmic fraction as a redox component which couldbe oxidised by TMAO or DMSO. Cytochrome c -556 was the only cytochrome species which co-fractionated with TMAO/DMSO reductase following gel filtration of a post-chromatophore supernatant produced after French presstreatment of intact cells. The mid-point redox potential (pH 7.6) of cytochrome c -556 was + 105 mV ( n = 1). It is suggested that TMAO/DMSO reductase and cytochrome c -556 form a structural and functional association in the periplasm of Rhodobacter capsulatus .

Inhibitory effects of myxothiazol and 2-n-heptyl-4-hydroxyquinoline-N-oxide on the auxiliary electron transport pathways of Rhodobacter capsulatus

The effects of various electron transport inhibitors upon the rates of reduction NO3-, dimethyl sulphoxide (DMSO) and N2O in anaerobic suspensions of Rhodobacter capsulatus have been studied. A new method for the determination of the rates of reduction of these auxiliary oxidants in intact cells is presented, based on the proportionality observed between the concentration of oxidant and the duration of the electrochromic carotenoid bandshift. For NO3-and N2O good agreement was found between rates of reduction determined using electrodes and those determined by the electrochromic method.Myxothiazol and antimycin A had no effect on the rates of reduction of NO3-and DMSO suggesting that the cytochrome b/c1complex is not involved in electron transport to these oxidants. 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) inhibited at two sites, one within the cytochrome b/c1complex and the other on the nitrate reducing pathay, but had no effect on electron transport to N2O or DMSO. In both intact cells and cell free extracts, HOQNO had no effect on the nitrate dependent re-oxidation of reduced methylviologen (MVH2), a direct electron donor to nitrate reductase.Our data are consistent with a branch point for the auxiliary electron transport pathways at the level of the ubiquinone pool.

Biochemistry and molecular biology of nitrification.

Publisher Summary The biochemistry and molecular biology of nitrification are poorly understood, almost certainly related to the difficult problem of growing large enough quantities of cells from which to prepare vesicular membranes and purified proteins. This chapter explains the biochemistry and molecular biology of nitrification. Nitrosomonas and Nitrobacter depend on a chemiosmotic mechanism of energy transduction. Many of the special biochemical features of Nitrosomonas and Nitrobacter need to be understood in the context of the ability of the electron transport system to catalyze reversed electron transfer. The demonstration of H_ pumping by intact cells fed with electrons from the nonphysiological donor ascorbate can be taken as support for the H_ pumping activity. The genome sequence clearly shows two reading frames, designated NorA and NorB on the basis of earlier partial sequence information. Bioenergetic arguments have suggested a location at the cytoplasmic surface, but immunolabeling studies have indicated the opposite. The oxidation of ammonia to NO 2 − by Nitrosomonas is not a straightforward process. The idea that ubiquinol provides electrons for the ammonia mono-oxygenase is supported by the fact that partially purified preparations of the enzyme can use duroquinol as electron donor.

Isolation of transposon Tn5 insertion mutants of Rhodobacter capsulatus unable to reduce trimethylamine-N-oxide and dimethylsulphoxide

Abstract1) Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata) strain 37b4 was subjected to transposon Tn5 mutagenesis. 2) Kanamycin-resistant transconjugants were screened for their inability to reduce trimethylamine-N-oxide (TMAO) as judged by the lack of alkali production during anaerobic growth on plates containing glucose as carbon source and cresol red as pH indicator. 3) Of 6 mutants examined, all were found to have considerably decreased levels of methylviologen-dependent TMAO reductase activity and dimethylsulphoxide (DMSO) reductase activity. 4) Periplasmic fractions of one of these mutants (DK9) and of the parent strain were subjected to sodium dodecylsulphate polyacrylamide gel electrophoresis. The gels were stained for TMAO-reductase and DMSO-reductase. With the wild-type strain, only a single polypeptide band, Mr=46,000, stained for TMAO and DMSO reductase activity. In mutant DK9 this band was not detectable. 5) In contrast to the parent strain, harvested washed cells of mutant DK9 were unable to generate a cytoplasmic membrane potential in the presence of TMAO or DMSO under dark anaerobic conditions. 6) In contrast to the parent strain, DK9 was unable to grow in dark anaerobic culture with fructose as the carbon source and TMAO as oxidant.

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains. The enzyme in strain BK5 is shown to be both functionally and structurally related to the nitrate reductase of Paracoccus denitrificans and Escherichia coli.

Dimethylsulphoxide and trimethylamine-N-oxide as bacterial electron transport acceptors: use of nuclear magnetic resonance to assay and characterise the reductase system in Rhodobacter capsulatus

Nuclear magnetic resonance is established as a sensitive and specific method for following the reduction of dimethylsulphoxide and trimethylamine-N-oxide by bacteria. Using this method it has been shown that cells of Rhodobacter capsulatus reduce both dimethylsulphoxide and trimethylamine-N-oxide at linear rates at all concentrations of these acceptors that can be conveniently detected during a continuous assay. The rate of reduction of trimethylamine-N-oxide was eightfold higher than the rate of dimethylsulphoxide reduction. An upper limit of approximately 0.1 mM may be placed upon the apparent Km value for each acceptor, but the value for dimethylsulphoxide is deduced to be lower than that for trimethylamine-N-oxide on the basis of the strong inhibitory effect of the former on the reduction of the latter. Reduction of trimethylamine-N-oxide by Rb. capsulatus was inhibited by illumination and by oxygen, but only the former effect was relieved following dissipation of the proton electrochemical gradient across the cytoplasmic membrane. Rotenone inhibited the reduction of trimethylamine-N-oxide whereas myxothiazol did not, consistent with a pathway of electrons to the reductase from NADH dehydrogenase that does not involve the cytochrome bc1complex.

The Prokaryotic Nitrate Reductases

This chapter reviews the structural organization and bioenergetics of the four prokaryotic NO 3 reductases and the eukaryotic enzyme and explores the possible mechanisms of NO 3 transport. The membrane-bound NO 3 − reductase with the active site facing the cytoplasm is usually a three-subunit enzyme composed of NarGHI. The Mo ion of NarG is coordinated by an aspartate ligand provided by the polypeptide chain. Adjacent to the structural genes of NarGHI in many denitrifying bacteria are one or two members of genes encoding transport proteins generally known as NarK family proteins. Where respiratory NO 3 reduction has been identified in Archaea, it is predicted to take place in a catalytic subunit with a twin arginine-dependent translocase (TAT) signal peptide, which may serve to export folded redox proteins across the cytoplasmic membrane. Periplasmic NO 3 reductases (Nap) are also linked to quinol oxidation in respiratory electron transport chains but do not transduce the free energy in the QH 2 NO 3 coupled into an H motive force. Bioinformatic analyses reveal that the Nap is phylogenetically widespread in proteobacteria, but detailed biochemical and spectroscopic studies have been restricted to enzymes from relatively few species. Some fungi have the capacity to reduce NO 3 as part of a denitrification process and here the NO 3 reductase is located in the mitochondrial membrane and is likely to emerge as being a prokaryotic pNar or nNar type.