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

Abstract

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...

Respiratory Properties of Cytochrome‐c‐Deficient Mutants of Azotobacter vinelandii

“…Cytochromes of the c-type are required for the oxidation of artificial electron donors such as reduced N,N,N ',N'-tetramethyl-p-phenylenediamine [Ph(NMe&]. This study could not demonstrate a physiological role for the c-type cytochromes which further supports the idea that the minor Ph(NMe2)z-oxidizing pathway of the electron transport chain may be independent of the major pathway terminated by cytochrome d.In a previous study, we reported on the isolation and characterization of a Ph(NMe2)2-oxidase-negative mutant of Azotohacter vinelundii [1]. This mutant appeared to be the first reported respiratory mutant for A .…”

“…It has been generally concluded that the minor reduced Ph(NMe& branch of the Azotohucter vineIundii respiratory chain carries only a small portion of the overall electron flow [1,. Despite a significant increase in the levels of components of the branch oxidizing reduced Ph(NMe2)z under oxygenlimiting conditions, concomitant increases in electron flow or efficiency of energy conservation have not been completely demonstrated.…”

“…To answer this possible criticism, mutants deficient in the c-type cytochromes were obtained. Since the c-type cytochromes appear to be required as electron acceptors for reduced Ph(NMe2)2 and also since these cytochromes are required for electron flow to the minor oxidase [1,5,12,15], these mutants should clearly be defective in the reduced Ph(NMe& oxidase pathway of electron transport. The results of this study suggest that the c-type cytochromes as well as the energy obtained from proton translocation at site I11 are not essential for growth or for nitrogen fixation.…”

“…In a previous study, we reported on the isolation and characterization of a Ph(NMe2)2-oxidase-negative mutant of Azotohacter vinelundii [1]. This mutant appeared to be the first reported respiratory mutant for A .…”

Cloning and mutagenesis of genes encoding the cytochrome bd terminal oxidase complex in Azotobacter vinelandii: mutants deficient in the cytochrome d complex are unable to fix nitrogen in air

“…The respiratory chain of A. vinelandii terminates in two branches with cytochromes o and d, respectively, as terminal oxidases (16). Mutants defective in the branch terminated by cytochrome o are unaffected in aerobic N2 fixation (16,17).…”