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

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)2. 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)2 as substrate. The oxidase-negative properties of mutant AV-11 appear to be associated with an inability of the terminal oxidases cytochromes o and a1 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)2 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 μmol oxygen consumed × min−1× mg protein−1; Km (O2) = 18 μM]. The respiratory kinetics exhibited for oxidation of reduced Ph(NMe2)2 via the oxidases cytochrome o and a1 could only be determined for strain AV-OP (V= 0.7 μmol O2× min−1× mg protein−1; Km= 3.1 μM). 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 a1, we suggest that for the Ph(NMe2)2-oxidase-negative mutants, cytochrome d is the only functional oxidase.