Synthesis and sideedness of membrane-bound respiratory nitrate reductase (EC1.7.99.4) in <i>Escherichia coli</i> lacking cytochromes

Kemp, M. B.; Haddock, Bruce A.; Garland, P. B.

doi:10.1042/bj1480329

Cited by 44 publications

(25 citation statements)

References 17 publications

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“…1B obliges us to leave open the question of the exposure of cytochrome b NO~ on the cytoplasmic surface of the cytoplasmic membrane. These conclusions are supported by the results of preliminary duplicate experiments using [ass] diazobenzene sulphonate in place of ~2SI/lactoperoxidase, by experiments using permeant and non-permeant reductants of nitrate reductase [14] and antibody agglutination studies (unpublished). The nitrate respiration complex therefore spans the cytoplasmic membrane of E. coli.…”

Section: Resultsmentioning

confidence: 65%

A Transmembrane location for the proton‐translocating reduced ubiquinone→ nitrate reductase segment of the respiratory chain of Escherichia coli

Boxer

Clegg

1975

FEBS Letters

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Section: Resultsmentioning

confidence: 65%

A Transmembrane location for the proton‐translocating reduced ubiquinone→ nitrate reductase segment of the respiratory chain of Escherichia coli

Boxer

Clegg

1975

FEBS Letters

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“…This was generated electrochemically (Thorneley, 1974) and its concentration estimated spectrophotometrically (after anaerobic dilution to minimise the effects of radical dimerization; E~~~~,~, 7.4 mM-' cm-', Kemp et al, 1975). Procedure C1 corresponds to reduction of the enzyme by approximately 100 pM BV" (i.e.…”

Section: Specific Procedures For Generating Epr Signals and For Testimentioning

confidence: 99%

Multiple States of the Molybdenum Centre of Dimethylsulphoxide Reductase from Rhodobacter Capsulatus Revealed by EPR Spectroscopy

Bennett

Benson

McEwan

et al. 1994

European Journal of Biochemistry

137

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The dimethylsulphoxide reductase of Rhodobacter capsulatus contains a pterin molybdenum cofactor molecule as its only prosthetic group. Kinetic studies were consistent with re-oxidation of the enzyme being rate limiting in the turnover of dimethylsulphoxide in the presence of the benzyl viologen radical. EPR spectra of molybdenum(V) were generated by reducing the highly purified enzyme under a variety of conditions, and with careful control it was possible to generate at least five clearly distinct EPR signals. These could be simulated, indicating that each corresponds to a single chemical species. Structures of the signal-giving species are discussed in light of the EPR parameters and of information from the literature. Three of the signals show coupling of molybdenum to an exchangeable proton and, in the corresponding species, the metal is presumed to bear a hydroxyl ligand. One signal with g,, 1.96 shows a very strong similarity to a signal for the desulpho form of xanthine oxidase, while two others with g,, values of 1.98 show a distinct similarity to signals from nitrate reductase of Escherichia coli. These data indicate an unusual flexibility in the active site of dimethylsulphoxide reductase, as well as emphasising structural similarities between molybdenum enzymes bearing different forms of the pterin cofactor. Interchange among the different species must involve either a change of coordination geometry, a ligand exchange, or both. The latter may involve replacement of an amino acid residue co-ordinating molybdenum via 0 or N, for a cysteine co-ordinating via S. Since the two signals with g,, 1.96 were obtained only under specific conditions of reduction of the enzyme by dithionite, it is postulated that their generation may be triggered by reduction of the pteridine of the molybdenum cofactor from a dihydro state to the tetrahydro state.Enzymes that depend on the pterin molybdenum cofactor (Rajagopalan, 1991) are widely distributed, have a variety of important roles in different organisms and have been extensively studied in many laboratories (for recent reviews, see Spiro, 1985;Bray, 1988;Solomonson and Barber, 1990;Wootton et al., 1991 ;Enemark and Young, 1993). Detailed structural information from X-ray crystallography is not yet available on any of these enzymes (however, see Romao et al., 1993a,b). All but one of the enzymes contain, in addition to molybdenum, redox centres such as flavin, haem or ironsulphur. These complicate spectroscopic investigations and, in particular, their strong absorption in the ultraviolethisible region effectively masks the rather weak absorption of the molybdenum chromophore. Nevertheless, other spectroscopic methods have provided important insights into the local structure around the molybdenum atom. EPR spectroscopy has been very important in this context and has been supplemented particularly by extended X-ray absorption fine structure (EXAFS) and to a lesser extent by magnetic circular Abbreviations. MCD, magnetic circular dichroism ; EXAFS, extended X-ray absorpt...

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“…The hem-mutant of E. coli studied by MacGregor (15) formed excess cytoplasmic enzyme when grown without ALA, indicating that the heme deficiency was influencing formation and integration of the enzyme into the membrane. In contrast, the mutant used by Kemp et al had less nitrate reductase activity when grown without supplement, and no difference was found in distribution of the enzyme between membrane and cytoplasm (11). Yet another pattern was observed with a hemmutant of Proteus mirabilis, which lacked nitrate reductase activity under conditions of heme deficiency (5).…”

Section: Downloaded Frommentioning

confidence: 88%

“…Reconstruction of b-and o-type cytochromes in deficient membranes was achieved with hemin but only in the presence of adenosine 5'-triphosphate. The E. coli mutants also differ from staphylococci in that nitrate reductase activity with physiological reductants is not restored by treatment of deficient membranes with hemin, and such activity is not restored to intact cells by ALA (11). Presumably the apo-protein of cytochrome b, is not formed, or it is degraded, when the heme prosthetic group is unavailable.…”

Section: Discussionmentioning

confidence: 99%

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Nitrate reductase activity in heme-deficient mutants of Staphylococcus aureus

Burke¹,

Lascelles²

1976

J Bacteriol

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Mutants H-14 and H-18 of Staphylococcus aureus require hemin for growth on glycerol and other nonfermentable substrates. H-14 also responds to delta-aminolevulinate. Heme-deficient cells grown in the presence of nitrate do not have lactate-nitrate reductase activity but gain this activity when incubated with hemin in buffer and glucose. Lactate-nitrate reductase activity is also restored to the membrane fraction from such cells by incubation with hemin and dithiothreitol; addition of adenosine 5'-triphosphate has no effect upon the restoration. Cells grown with nitrate in the absence of hemin have two to five times more reduced benzyl viologen-nitrate reductase activity than do those grown with hemin. The activity increases throughout the growth period in the absence of hemin, but with hemin present enzyme formation ceases before the end of growth. There was no evidence of enzyme destruction. The distribution of nitrate reductase activity between membrane and cytoplasm was similar in cells grown with and without hemin; 70 to 90% was in the cytoplasm. It is concluded that heme-deficient staphylococci form apo-cytochrome b, which readily combines in vitro with its prosthetic group to restore normal function. The avaliability of the heme prosthetic group influences the formation of nitrate reductase.

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Synthesis and sideedness of membrane-bound respiratory nitrate reductase (EC1.7.99.4) in Escherichia coli lacking cytochromes

Cited by 44 publications

References 17 publications

A Transmembrane location for the proton‐translocating reduced ubiquinone→ nitrate reductase segment of the respiratory chain of Escherichia coli

A Transmembrane location for the proton‐translocating reduced ubiquinone→ nitrate reductase segment of the respiratory chain of Escherichia coli

Multiple States of the Molybdenum Centre of Dimethylsulphoxide Reductase from Rhodobacter Capsulatus Revealed by EPR Spectroscopy

Nitrate reductase activity in heme-deficient mutants of Staphylococcus aureus

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