The use of 3,3''-diaminobenzidine as a biochemical and histochemical electron donor for the terminal oxidase site in Azotobacter Vinelandii Strain O



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Both Azotobacter vinelandii whole cells and the R[subscript 3] electron transport particle (ETP) were examined for their capability of oxidizing 3,3'-diaminobenzidine (DAB), a dye suspected of serving as an electron donor at the terminal oxidase site. The DAB terminal oxidase activity was quantitatively compared to the N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) oxidation reaction which is known to carry out an identical type of reaction at this same oxidase site. The DAB oxidase as well as the TMPD oxidase proved to be almost "identically" sensitive to the classical cytochrome oxidase inhibitors, KCN, NaN[subscript 3], and NH[subscript 2]OH. A standardized manometric assay system was developed for the DAB oxidase reaction that defines (a). the optimal concentration of ascorbate that was needed to chemically reduce DAB, (b). established the concentration of DAB that allowed for maximal activity as well as the optimal pH for DAB oxidase reaction. Difference spectra studies showed that both DAB and TMPD perturbated the absorption characteristics of cytochromes c[subscript 4] + c[subscript 5] and cytochrome o, suggesting that these are the terminal cytochrome oxidase components which are responsible for carrying out both DAB and TMPD oxidation in the A. vinelandii electron transport system. It was also possible to confirm that 1 mole of DAB reacted with 1 mole of oxygen according to the molecular stoichiometry predicted by the chemical oxidation reaction. The molecular stoichiometry for TMPD oxidation could not be confirmed indicating that this reaction, although used universally for studying cytochrome oxidase activity (particularly in mammalian mitochondrial systems) is not really understood. It was also possible to use DAB to histochemically localize the terminal oxidase site in Azotobacter vinelandii strain O. The site of DAB oxidase activity appears to be almost exclusively on the cytoplasmic membrane, while the extensive inner network of membranes, found within this organism, appear to be "relatively" free of DAB deposits generated by the cytochrome oxidase activity. This histochemical study represents the first successful attempt at specifically localizing the terminal cytochrome oxidase site in a bacterium.



Biology, Biochemistry