William J. Payne

Regulation of the hexaheme nitrite/nitric oxide reductase of Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli: A mass spectrometric study

Dissimilatory nitrite reduction, carried out by hexaheme proteins, gives ammonia as the final product. Representatives of this enzyme group from 3 bacterial species can also reduce NO to either ammonia or N2O. The redox regulation of the nitrite/nitric oxide activities is discussed in the context of the denitrifying pathway.

A Halophilic Denitrifier, Bacillus halodenitrificans sp. nov.

A new halotolerant denitrifier, which was isolated from a solar saltern by enrichment culture in liquid medium supplemented with 1.06 M (9%) NaNO3, grew optimally in media containing 0.5 to 1.35 M NaCl, survived and multiplied in media ranging in salinity from 0.35 to 4.25 M NaCl, and tolerated high nitrite ion concentrations, as well as high nitrate ion concentrations. The salt requirement could be provided by 1 M KNO3 or KCl instead of NaCl. For this nonfermentative organism, nitrate and nitrite were the only electron acceptors tested that supported anaerobic growth on a complex medium. Washed cells reduced both nitrate and nitrite at significant rates. The isolate lacked a nitrous oxide reductase activity, utilized a variety of substrates as carbon and energy sources, and required both growth factors and organic (reduced) sulfur. Ammonia served as a nitrogen source for growth, but nitrate did not. Despite the failure of the organism to sporulate, assignment to the genus Bacillus appeared to be consistent with results of cell constituent analyses and partial 16S ribosomal ribonucleic acid sequencing. We propose the name Bacillus halodenitrificans for this organism. A type culture has been deposited with the American Type Culture Collection, Rockville, Md., as strain ATCC 49067.

Isolation and Preliminary Characterization of a Soluble Nitrate Reductase from the Sulfate Reducing Organism Desulfovibrio desulfuricans ATCC 27774

Desulfovibrio desulfuricans ATCC 27774 is a sulfate reducer that can adapt to nitrate respiration, inducing the enzymes required to utilize this alternative metabolic pathway. Nitrite reductase from this organism has been previously isolated and characterized, but no information was available on the enzyme involved in the reduction of nitrate. This is the first report of purification to homogeneity of a nitrate reductase from a sulfate reducing organism, thus completing the enzymatic system required to convert nitrate (through nitrite) to ammonia. D. desulfuricans nitrate reductase is a monomeric (circa 70 kDa) periplasmic enzyme with a specific activity of 5.4 K(m) for nitrate was estimated to be 20 microM. EPR signals due to one [4Fe-4S] cluster and Mo(V) were identified in dithionite reduced samples and in the presence of nitrate.

Suppression by nitrate of enzymatic reduction of nitric oxide.

Summary We found that the entire array of enzymes in Pseudomonas perjectomarinus that account for reduction of nitrate to nitrogen, including that which reduces nitrous oxide to nitrogen, was synthesized in 40, but not 35, min of incubation in a complete medium containing nitrate. Nitrite accumulated during the first 5-6 hr of culture despite the fact that we could demonstrate enzymes in crude cell-free extracts that reduced nitrite, nitric oxide, and nitrous oxide. To explain this, we found that nitrate suppressed the activity of nitric oxide reducing enzyme in crude extracts, as well as that in a fraction containing none of the other relevant reducing systems.

THE DENITRIFYING NITRITE REDUCTASE OF BACILLUS HALODENITRIFICANS

Denitrifying nitrite reductase was purified from Bacillus halodenitrificans, a newly isolated moderate halophile, and identified as a dimeric nonheme copper protein with a molecular mass of 82 kDa comprising two identical 40-kDa subunits. Unlike its counterparts in other denitrifiers, this enzyme is firmly bound in the cytoplasmic membrane, a characteristic that may be related to the Gram-positive nature of this bacillus (and thus lack of a periplasmic space). The enzyme functioned most effectively at pH 6, and was activated by elevated salt concentrations, a property shared by the copper nitrite reductase from another, but nonhalophilic, denitrifier, Achromobacter cycloclastes. In contrast, high salt activation was not exhibited by cytochrome cd1 nitrite reductases from either nonhalophilic Thiobacillus denitrificans or halophilic Paracoccus halodenitrificans. The enzyme was rich in aspartate/asparagine and methionine residues. Electron paramagnetic resonance spectroscopy revealed both type 1 and type 2 copper in the protein. The physiological electron donor that transfers electrons to the copper nitrite reductase from A. cycloclastes, a small blue copper protein, did not transfer electrons to the copper nitrite reductase of B. halodenitrificans.

On the purification of nitrite reductase from Thiobacillus denitrificans and its reaction with nitrite under reducing conditions

Abstract Nitrite reductase (cytochrome cd ) from T. denitrificans has been crystallized in high yield in three simple and rapid steps. The spectral absorption ratio at 408 to 280 nm was 1.52. Light absorption spectra in the oxidized and reduced states were virtually identical to those of nitrite reductase from P. aeruginosa . EPR spectroscopy of nitrite reductase at 12° showed a low-spin ferric heme resonance with g-values at 2.52, 2.45 and 1.73 assigned to the d-heme. Reaction of nitrite reductase with nitrite in the presence of the reducing systems [(ascorbate + PMS) or sulfide] resulted in the formation of nitric oxide (confirmed by gas chromatography) which reacted with both c - and d -hemes of nitrite reductase yielding an EPR-detectable enzyme-NO complex with g-values at 2.07, 2.04 and 1.99 and a 14N hyperfine splitting constant of 22.5 gauss. The amount of nitric oxide produced enzymatically with sulfide as electron donor was only 5% of that found when ascorbate plus PMS served as reductant. To our knowledge the detection of the unique enzyme-NO complex is the first definitive EPR evidence for the mandatory liganding of nitric oxide with pure nitrite reductase during nitrite reduction.

Electron paramagnetic resonance studies on the nature of hemoproteins in nitrite and nitric oxide reduction.

Abstract Nitrite and nitric oxide reductases and c -type cytochromes were isolated from nitrate-grown Pseudomonas perfectomarinus . EPR measurements indicate the direct participation of c -type cytochromes in the reduction of nitrite to nitric oxide. The EPR resonance of heme-NO complexes is observed with g -values at 2.03 and 2.10 with 14 N hyperfine splitting of 16.2 Gauss. Quantitative EPR measurements indicate that all the c -type cytochromes involved in nitrite reduction are in the heme-NO complex forms and that these complexes account for about 15% of the total nitric oxide observed. A different c -type cytochrome was found to be involved in the reduction of NO to N 2 O with the major g -value of the heme-NO complex at 2.06. A time-dependent broadening of EPR was noted.

Localization and specificity of cytochromes and other electron transfer proteins from sulfate-reducing bacteria

Recently data have accumulated concerning the electron transfer chains of sulfate-reducing bacteria in general and of the genus Desulfovibrio in particular. Because of the ever growing number of newly discovered individual redox proteins, it has become essential to try to assign them to physiologically relevant chains. This work presents some new data concerning the localization of these proteins within the bacterial cell and the specificity of electron transfer between the three types of hydrogenases which have been found so far in Desulfovibrio, namely the iron-only, the iron-nickel and the iron-nickel-selenium enzymes. The iron-only hydrogenase reduces cytochromes which have bis-histidinyl heme ligation or histidinyl-methionyl heme ligation. In contrast, the iron-nickel and iron-nickel-selenium hydrogenases cannot reduce cytochromes having a His-Met heme ligation, but are very active toward the cytochromes having a bis-histidinyl ligand. This observation has been used to demonstrate that the tetraheme cytochrome c3 can exchange electrons with the monoheme cytochrome c553. No clear specificity has been established for the reaction of hydrogenases toward the hexadecaheme cytochromes from either D vulgaris or D gigas.

Nitrite reduction in Rhizobium hedysarï strain HCNT 1

Rhizobium “hedysari” strain HCNT 1 rapidly reduced nitrite to N2O, only slowly reduced nitrate to nitrite and did not exhibit nitrous oxide reductase activity. Nitrite reduction in this rhizobium strain may be a detoxification mechanism for conversion of nitrite, which inhibits O2 uptake, to non-toxic N2O. Concentrations of nitrite as small as 3 μM diminished O2 uptake in whole cells. The bacterium did not couple energy conservation with nitrate or nitrite reduction. Cells neither grew anaerobically at the expense of these nitrogen oxides nor translocated protons during reduction of nitrite. Induction of nitrite reductase activity was not a response to the presence of nitrate or nitrite, but occurred instead when the O2 concentration in culture atmospheres fell to <16.5% of air saturation. Sensitivity of cytochrome o, which is synthesized only in cells grown under O2-limited conditions, may account for the toxicity of nitrite in strain HCNT 1.

Some observations on the physiology ofPseudomonas natriegens nov. spec.

SummaryPseudomonas natriegens, nov. spec. which requires Na+ for growth, has been found to produce a considerable quantity of acid by the dissimilation of glucose in aerobic cultures. The products of glucose catabolism have been identified as CO2 and acetic, pyruvic and lactic acids. Acid production is very rapid as well in broth cultures containing sodium glucuronate, but not in cultures containing galacturonate.Growth of this bacterium was not inhibited by saturating quantities of 2,4-diamino-6,7-diisopropyl pteridine. This property indicates that the isolate is more likely to be appropriately placed in the genusPseudomonas than in the genusVibrio.Induction of enzymes in resting cells for the oxidation of glucuronate was inhibited by chloramphenicol added at various intervals during the first 2 hrs of incubation.