Open AccessRespiratory‐Chain Characteristics of Mutants of Azotobacter vinelandii Negative to Tetramethyl‐ p ‐phenylenediamine Oxidase
Author(s) -
HOFFMAN Paul S.,
MORGAN T. Vance,
VARTANIAN Daniel V.
Publication year - 1979
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1979.tb02029.x
Subject(s) - azotobacter vinelandii , biochemistry , mutant , chemistry , alternative oxidase , respiratory chain , oxidase test , azotobacter , oxygen , cytochrome c oxidase , cytochrome , ferricyanide , enzyme , biology , bacteria , nitrogenase , organic chemistry , genetics , nitrogen fixation , nitrogen , gene
Mutants of Azotobacter vinelandii negative to N,N,N′,N′ ‐tetramethyl‐ p ‐phenylenediamine [Ph(NMe 2 ) 2 ] oxidase were obtained after mutagenesis by screening colonies with the ‘oxidase reagent’ Ph(NMe 2 ) 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 N 2 ‐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(NMe 2 ) 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 a 1 to reoxidize cytochromes c 4 + c 5 in membrane particles. Cytochrome c 4 + c 5 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 c 4 + c 5 of mutant AV‐11 are readily reduced by reduced Ph(NMe 2 ) 2 and studies on partially purified cytochromes c 4 and c 5 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 ; K m (O 2 ) = 18 μM]. The respiratory kinetics exhibited for oxidation of reduced Ph(NMe 2 ) 2 via the oxidases cytochrome o and a 1 could only be determined for strain AV‐OP ( V = 0.7 μmol O 2 × min −1 × mg protein −1 ; K m = 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 c 4 + c 5 and the oxidases cytochromes o and a 1 , we suggest that for the Ph(NMe 2 ) 2 ‐oxidase‐negative mutants, cytochrome d is the only functional oxidase.