Respiration‐linked proton translocation in <i>Azotobacter vinelandii</i>

Downs, Andrew J.; Jones, Colin W.

doi:10.1016/0014-5793(75)80414-5

Cited by 32 publications

(33 citation statements)

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“…The oxidation of artificial electron donors such as ascorbate-N,N,N',N'-tetramethyl-pphenylene diamine [Ph(NMe,),] proceeds via c-type cytochromes to the o, u1 oxidase [3,11 -131 and is readily inhibited by cyanide [3,9: 141. Sites of cncrgy conservation occur at the level of NADH dchydrogenase, hydrogenase, the ubiquinone region [4,9,7,16,17,33]. Recent studies diminish the possibility that the cytochrome c-al, o branch is involved in the energy conservation cfficicncy under non-oxygen-limiting growth conditions [11,12.13:15].…”

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Respiratory mutants of Azotobacter vinelandii with elevated levels of cytochrome d

et al. 1984

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A method is described for the isolation of respiratory mutants of Azotobacter cinelundii with incrcascd amounts of cl-type cytochrome by selecting for the inability to reduce tctrazolium red. Five stable mutants were obtained that had six-fold higher levels of cytochrome d, increased amounts of b-type and lower amounts of o-type and c-type cytochromes than the wild-type strain. Spectral alterations in cytochrome al were also observed in the mutants. N A D H and succinate oxidase activities of membrane particles were about two fold higher in the mutants compared to the wild-type strain. Ascorbate-N,N,N',N'-tetramethyl-p-phenylene diaminc oxidase activity was barely detectable in membranc particles of the mutants. These results are consistent with an increasc in the amount of the cytochrome d oxidase branch and a decrease in the amount and activity of the cytochrome 0, a1 oxidase branch in the mutants. Growth rates under oxygen-excess conditions and respiratory-linked proton translocation ratios of the mutant and wild-type strains were similar as were the photochemical spectral and kinetic properties of cytochrome d.The respiratory chain of Azotobacter vinelundii has been extensively studied [l, 21. The components of the respiratory chain include highly active flavin-dependent NADH and malate dehydrogenases, hydrogenase, ubiquinone-8, and at least seven spectroscopically detectable cytochromes, b-560. [3,9: 141. Sites of cncrgy conservation occur at the level of NADH dchydrogenase, hydrogenase, the ubiquinone region [4,9,7,16,17,33]. Recent studies diminish the possibility that the cytochrome c-al, o branch is involved in the energy conservation cfficicncy under non-oxygen-limiting growth conditions [11,12.13:15].In addition to its role in energy conservation, it has been proposed that the high activity of the respiratory chain protects the oxygen-sensitive nitrogenase complex in Azotohacter from inactivation [18 -201. Respiratory protection of nitrogenase at higher dissolved oxygen tensions is provided by an increase in whole cell respiration rate which is accompanied by increases in N A D H oxidase activity and levels of cytochrome d [18 -201. The study of respiratory mutants of A . uinelandii has proven to be useful in determining the contribution of each branch of the respiratory chain in the oxidation of physiological substrates and in the efficiency of encrgy conservation. Biochemical studies of mutants unable to reoxidize Ph(NMe,), could not demonstrate a significant role for the MATbRIALS A N D METHODS Bacterial strainsThe wild-type strain AVOP of Azobacter rinelandii was used. Strains AV90 and AVZ 07 are Ph(NMe,),-oxidasenegative mutants of AVOP [I1 -131 (Hoffman and Dcr Vartanian, unpublished results). Mt.dia and conditions of cultiuatiorzEach strain was routinely grown in 1.5 1 of modified Burk's medium [21] with 2 '4 sucrose (wiv) without an added nitrogen source. Other ctrbon sourccs were added at a final concentration of 0.2 ,d (wlv) and nitrogen sources were added to givc a final combined niiro...

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Respiratory mutants of Azotobacter vinelandii with elevated levels of cytochrome d

et al. 1984

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“…Azotobacter membranes contain a rich variety of redox carriers, cytochromes, and dehydrogenases, arranged to support two or possibly three sites of respiration-driven proton translocation [5 -91. The cytochrome chain appears to be branched at cytochrome bl [7] and is terminated by three oxidases [5,7,10,11]. Inhibitor studies [5,7,11, 121 as well as studies by photochemical action spectral methods [8,10] suggest that the major flow of elecAbbreviation.…”

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“…The cytochrome chain appears to be branched at cytochrome bl [7] and is terminated by three oxidases [5,7,10,11]. Inhibitor studies [5,7,11, 121 as well as studies by photochemical action spectral methods [8,10] suggest that the major flow of elecAbbreviation. Ph(NMe*)z, N,N, N', N'-Tetramethyl-p-phenylenediamine, also known as TMPD.…”

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Respiratory-Chain Characteristics of Mutants of Azotobacter vinelandii Negative to Tetramethyl-p-phenylenediamine Oxidase

Hoffman¹,

Morgan²,

Vartanian³

1979

Eur J Biochem

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Mutants of Azotobacter vinelandii negative to N, N, N', N'-tetramethyl-p-phenylenediamine[Ph(NMe2)2] oxidase were obtained after mutagenesis by screening colonies with the 'oxidase reagent' Ph(NMe2)z. These mutants appear to be the first respiratory mutants reported for Azotobacter. Characterization of one of these mutants (AV-11) revealed similar growth rates under N2-fixing conditions, comparable P/O ratios (about 0.6, NADH) and H/O quotients (about 4), and similar respiration rates as the parent strain AV-OP. No oxygen consumption or net synthesis of ATP could be demonstrated with phosphorylating membranes of mutant AV-11 using reduced Ph(NMe2)z as substrate. The oxidase-negative properties of mutant AV-11 appear to be associated with an inability of the terminal oxidases cytochromes o and al to reoxidize cytochromes c4 + c5 in membrane particles. Cytochrome c4 + c5 of mutant AV-11 could not be reoxidized by normal procedures (bubbling with oxygen or with 0.05 mM ferricyanide). These could only be reoxidized by excess ferricyanide (10-20 mM). Oxidized cytochromes c4 + c5 of mutant AV-11 are readily reduced by reduced Ph(NMe2)Z and studies on partially purified cytochromes c4 and c5 showed no unusual properties.A comparison of the respiratory kinetics for membrane particles of strains AV-11 and AV-OP showed no differences in the oxidation of NADH or malate via cytochrome oxidase d [ V = 3.2 pmol oxygen consumed x min-' x mg protein-'; K , ( 0 2 ) = 18 pM]. The respiratory kinetics exhibited for oxidation of reduced Ph(NMe& via the oxidases cytochrome o and a1 could only be determined for strain AV-OP (V = 0.7 pmol 0 2 x min-' x mg protein-'; K, = 3.1 pM). The very high V value observed for oxidation of cytochrome d (strains AV-11 and AV-OP) suggests that this oxidase is capable of handling the electron flow generated by the very active dehydrogenases. Since the respiratory chain of mutant AV-11 appears to be blocked between cytochromes c4 + c5 and the oxidases cytochromes o and al, we suggest that for the Ph(NMe2)2-oxidase-negative mutants, cytochrome d is the only functional oxidase.The respiratory chain of Azotobacter vinelandii has been extensively studied (for a partial review, see [l]) as has its role in protection of the oxygen-sensitive nitrogenase complex [2 -41. Azotobacter membranes contain a rich variety of redox carriers, cytochromes, and dehydrogenases, arranged to support two or possibly three sites of respiration-driven proton translocation [5 -91. The cytochrome chain appears to be branched at cytochrome bl [7] and is terminated by three oxidases [5,7,10,11]. Inhibitor studies [5,7,11, 121 as well as studies by photochemical action spectral methods [8,10] suggest that the major flow of elecAbbreviation. Ph(NMe*)z, N,N, N', N'-Tetramethyl-p-phenylenediamine, also known as TMPD.trons from the dehydrogenases are oxidized via cytochrome d. Cytochrome oxidases o and a1 terminate the minor branch and appear to be associated with cytochromes c4 + c5 [5,8,11,13]. The minor pathway contains a phosphor...

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“…In A. vinelandii (15), the electron transport chain is branched (4,16) and terminates at two terminal oxidases: the bo terminus, believed to be a high-affinity coupled oxidase that is induced during low oxygen growth conditions; and the cytochrome bd terminus, a low-affinity oxidase that is induced during high oxygen growth conditions (8,30). It is the cytochrome bd terminus, however, that supports the majority of electron flux in the organism (16) and is proposed to provide respiratory protection to nitrogenase (8,30).…”

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“…Other work, employing either whole cells or membrane preparations, has expanded upon the respiratory protection concept to suggest that the A. vinelandii cytochrome bd terminus of the electron transport chain is either uncoupled from or inefficiently coupled to oxidative phosphorylation (1,2,4). While this early work provided valuable information about the composition and sequence of the A. vinelandii electron transport chain components, the conclusions to be drawn from these indirect experiments pertaining to the coupling abilities of the oxidases cannot be firmly stated.…”

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Formation of pH and potential gradients by the reconstituted Azotobacter vinelandii cytochrome bd respiratory protection oxidase

Kolonay

Maier

1997

J Bacteriol

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To directly characterize the bioenergetic properties of the cytochrome bd terminating branch of the Azotobacter vinelandii electron transport chain, the purified cytochrome bd oxidase was reconstituted into a phospholipid environment consisting of phosphatidylethanolamine and phosphatidylglycerol (3:1). The average diameter of the proteoliposomes after extrusion through a polycarbonate membrane was 94 ؎ 4 nm. Initiation of respiration upon the addition of 20 M ubiquinone-1 to proteoliposomes loaded with the pH-sensitive dye pyranine resulted in an immediate alkalization of the vesicle lumen by an average pH change of 0.11 unit. This pH gradient was readily collapsed upon the addition of nigericin, carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrazone, gramicidin, Triton X-100, or 2-heptyl-4-hydroxyquinoline N-oxide (HQNO). Proteoliposomal respiration initiated in the presence of the potentiometric membrane dye rhodamine 123 caused the generation of a transmembrane potential; the potential was collapsed upon the addition of either valinomycin or HQNO. The formation of both pH and potential gradients during turnover demonstrates that the A. vinelandii cytochrome bd oxidase is coupled to energy conservation in vivo.

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Respiration‐linked proton translocation in Azotobacter vinelandii

Cited by 32 publications

References 23 publications

Respiratory mutants of Azotobacter vinelandii with elevated levels of cytochrome d

Respiratory mutants of Azotobacter vinelandii with elevated levels of cytochrome d

Respiratory-Chain Characteristics of Mutants of Azotobacter vinelandii Negative to Tetramethyl-p-phenylenediamine Oxidase

Formation of pH and potential gradients by the reconstituted Azotobacter vinelandii cytochrome bd respiratory protection oxidase

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