Quantitation of the TMPD Oxidase in Azotobacter vinelandii and other microorganisms



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It has been possible to quantitate the terminal oxidase reaction in a wide variety of physiologically diverse non-proliferating bacterial whole cells using a manometric N,N,N1,N1-tetramethyl-p-phenylenediamine (TMPD) oxidase assay. The assay, as developed for Asotobacter vinelandii, which possesses a very potent terminal oxidase, was extended to other bacteria to determine whether or not such a procedure would be taxonomically useful. The kinetics of TMPD oxidation by A. vinetandii whole cells was similar to that noted for the electron transport fraction, suggesting that reduced TMPD can penetrate whole cells and is readily oxidized by terminal oxidases that reside exclusively on the cytoplasmic membrane. The TMPD oxidase reaction was also employed for studies with A. vinetandtt respiratory deficient mutants as well as with the nutritionally induced pleomorphic cells. All TMPD oxidase QO2 values are expressed in [mu]l O2 consumed per hour per mg dry wt. For A. vinelandii whole cells the TMPD oxidase O2 value ranged from 1700 to 2000 and reflected the full measure of the high respiratory capacity usually noted for comparable A. vinetandii whole cells grown on acetate under nitrogen-fixing conditions. The TMPD oxidase activity in A. vinetandii was found to be affected by the growth conditions; sucrose grown cells exhibited much lower TMPD oxidase QO2 values than the cell suspension grown on acetate. [...]