Abstract
Azotobacter vinelandii cells readily oxidize the dye 3,3′-diaminobenzidine (DAB), which has been previously used as an electron donor for studies on the mitochondrial cytochromec oxidase reaction. The DAB oxidase activity inA. vinelandii cells was 10-fold lower than that noted for theN,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) oxidase reaction, which is commonly used to measure terminal oxidase activity both in bacteria and mitochondria. Analyses of cell-free extracts show that DAB oxidase activity is concentrated almost exclusively in theA. vinelandii membrane fractions, most notably in the “R3” electron transport particle (ETP). Oxidation studies, which employed both whole cells and the ETP fraction, show DAB oxidase activity to be markedly sensitive to KCN, NaN3, and NH2OH. A manometric assay system was developed which readily measured DAB oxidase activity in bacteria. Preliminary studies indicate that ascorbate-DAB oxidation inAzotobacter vinelandii measures terminal cytochrome oxidase activity in a manner similar to the TMPD oxidase reaction.
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Literature Cited
Ackrell, B. A. C., Jones, C. W. 1971. The respiratory system ofAzotobacter vinelandii. I. Properties of phosphorylating respiratory membranes. European Journal of Biochemistry20:22–28.
Ben-Hayyim, G. Z., Drechsler, J. G., Neumann, J. 1975. Diaminobenzidine, an electron donor to photosystem 1 and to photosystem 2 in chloroplasts. European Journal of Biochemistry52:135–141.
Burk, D. 1930. The influence of oxygen gas upon the organic catalysis of nitrogen fixation byAzotobacter. Journal of Physical Chemistry34:1195–1209.
Cammer, W., Moore, C. L. 1973. Oxidation of 3,3′-diaminobenzidine by rat liver mitochondria. Biochemistry12:2502–2509.
Chua, N. H. 1972. Photooxidation of 3,3′-diaminobenzidine by blue-green algae andChlamydomonas reinhardii. Biochimica et Biophysica Acta267:179–189.
Erickson, S. K., Diehl, H. 1973. The terminal oxidases ofAzotobacter vinelandii. Biochemical and Biophysical Research Communications50:321–327.
Jones, C. W., Redfearn, E. R. 1967. The cytochrome system ofAzotobacter vinelandii. Biochimica et Biophysica Acta143:340–353.
Jones, M. V. 1975. Physiological role for the membrane bound ascorbate-TMPD oxidase inPseudomonas putida. Archives of Microbiology102:275–279.
Jurtshuk, P., Jr., Aston, P. R., Old, L. 1967. Enzymatic oxidation of tetramethyl-p-phenylenediamine andp-phenylenediamine by the electron transport particulate fraction ofAzotobacter vinelandii. Journal of Bacteriology93:1069–1078.
Jurtshuk, P., Jr., McQuitty, D.N. 1976. Survey of oxidase-positive and-negative bacteria using a quantitative Kovacs oxidase test. International Journal of Systematic Bacteriology26:127–135.
Jurtshuk, P., Jr., McQuitty, D. N. 1976. use of a quantitative oxidase test for characterizing oxidative metabolism in bacteria. Applied and Environmental Microbiology31:668–679.
Jurtshuk, P., Jr., Marcucci, O. M., McQuitty, D. N. 1975. Tetramethyl-p-phenylenediamine oxidase reaction inAzotobacter vinelandii. Applied Microbiology30:951–958.
Jurtshuk, P., Jr., May, A. K., Pope, L. M., Aston, P. R. 1969. Comparative studies on succinate and terminal oxidase activity in microbial and mammalian electron-transport systems. Canadian Journal of Microbiology15:797–807.
Jurtshuk, P., Jr., Milligan, T. W. 1974. Quantitation of the tetramethyl-p-phenylenediamine oxidase reaction inNeisseria species. Applied Microbiology28:1079–1081.
Jurtshuk, P., Jr., Mueller, T. J., Acord, W. C. 1975. Bacterial terminal oxidases. CRC Critical Reviews in Microbiology3:399–468.
Jurtshuk, P., Jr., Mueller, T. J., McQuitty, D. N., Riley, W. H. 1978. The cytochrome oxidase reaction inAzotobacter vinelandii and other bacteria, pp. 99–121. In: Degn, H., Lloyd, D., Hill, G. C. (eds.), Functions of alternative terminal oxidases, vol. 49. Oxford: Pergamon Press.
Jurtshuk, P., Jr., Old, L. 1968. Cytochromec oxidation by the electron transport fraction ofAzotobacter vinelandii. Journal of Bacteriology95:1790–1797.
Postgate, J. 1971. Fixation by free-living microbes: Physiology, pp. 161–190. In: Postgate, J. R. (ed.), The chemistry and biochemistry of nitrogen fixation. London, New York: Plenum Press.
Reith, A., Schuler, B. 1972. Demonstration of cytochrome oxidase activity with diaminobenzidine. Biochemical and electron microscopic study. Journal of Histochemistry and Cytochemistry20:581–587.
Sekuzu, I., Takemori, S., Orii, Y., Okumuki, K. 1960. Studies on cytochromea. IV. Reaction of cytochromea with cytochromesc andc1. Biochimica et Biophysica Acta37:64–71.
Seligman, A. M., Karnovsky, M. J., Wasserkrug, H. L., Hanker, J. S. 1968. Nondroplet ultrastructural demonstration of cytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB). Journal of Cell Biology38:1–14.
Tyler, D. D., Estabrook, R. W. 1965. Electron and energy requirements for cytochromeb reduction during oxidation of tetramethyl-p-phenylenediamine. Biochemical and Biophysical Research Communications18:264–269.
Yang, T. Y., Jurtshuk, P., Jr. 1978. Purification and characterization of cytochromeo fromAzotobacter vinelandii. Biochimica et Biophysica Acta502:543–548.
Yang, T. Y., Jurtshuk, P., Jr. 1978. Studies on the red oxidase (cytochromeo) ofAzotobacter vinelandii. Biochemical and Biophysical Research Communications81:1032–1039.
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Jurtshuk, P., McQuitty, D.N. & Riley, W.H. Use of 3,3′-diaminobenzidine as a biochemical electron donor for studies on terminal cytochrome oxidase activity inAzotobacter vinelandii . Current Microbiology 2, 349–354 (1979). https://doi.org/10.1007/BF02602874
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DOI: https://doi.org/10.1007/BF02602874