Terminal oxidases of micrococcus denitrificans


Purification of the terminal oxidases of the facultative anerobe, M. denitrificans, was undertaken. The bacterium contains both a cytochrome c oxidase and a cyanide-insensitive DPNH oxidase. A procedure for purification of each of these enzymes is given. These procedures involve fractionation with ammonium sulfate, acetone, and calcium phosphate gel. By the use of the procedures given, cytochrome c oxidase can be purified seventeen-fold and the DPNH oxidase ninty-fold over the original cell-free extract. The cytochrome c oxidase was found to be associated with the particulate material of the cell, had an optimum activity at pH 7.0 to 7.4, and was not affected by aluminum, magnesium, or manganous ion. The enzyme was completely inhibited by cyanide and carbon monoxide but only 65% by azide. The cytochrome c oxidase oxidized reduced mammalian cytochrome c directly. Oxidative phosphorylation was demonstrated during oxidation of reduced mammalian cytochrome c by cell-free extracts of the bacteria. The absorption maxima of the reduced spectrum of the partially purified cytochrome c oxidase were at 420, 522, and 554 mμ., and the maxima of the difference spectrum were at 427, 522, and 551 mμ. The DPNH oxidase appears to be a soluble flavoprotein. An active DPNH oxidase preparation which was inactivated by ammonium sulfate fractionation, could be reactivated by addition of flavin mononucleotide; flavin adenine dinucleotide restored only partial activity. The Michaelis constant with respect to DPNH of the partially purified DPNH oxidase was found to be 3.42 x 10^-6 moles/liter. The activation energy of the DPNH oxidase was determined and found to be 14,900 cals/mole. The oxygen uptake of a cell-free extract of M. denitrificans functioning as a cytochrome c oxidase and as a DPNH oxidase, was 7.54 u liters/minute and 3.52 u liters/minute respectively. The cytochrome c oxidase appears to be the primary terminal oxidase, however, the DPNH oxidase does make a significant contribution to the aerobic respiration of the bacterium. The participation of the bacterial cytochrome c oxidase and DPNH oxidase in the aerobic respiration of the bacterium is discussed.



College and Department

Chemistry and Biochemistry



Date Submitted


Document Type





Micrococcus dentrificans, Terminal oxidases of



This document is currently not available here.