Cytochemistry of Membrane-Associated Cytochrome Oxidase in Azotobacter Vinelandii

Abstract

Young, vegetative cells of the nitrogen-fixing bacterium, Azotobacter vinelandii, contain invaginations of the cytoplasmic membrane. These typical procaryotic cells are characterized by the absence of both a nuclear membrane and organelles such as Golgi bodies and mitochondria (Fig. 1). Although mitochondria are the organelles responsible for electron transport and respiratory operations in eucaryotic organisms, the electron transport function of procaryotic cells is attributed to the membranous structures. In the present study an attempt was made to visualize the site of cytochrome oxidase activity using methods of cytochemistry and electron microscopy.The classical method for the demonstration of cytochrome oxidase activity is the Nadi reaction; the two reagents of this reaction are 1-naphthol and p-phenylenediamine. A substituted p-phenylenediamine, N-benzyl-p-phenylenediamine (BPDA), was used successfully by Seligman et al in the demonstration of cytochrome oxidase activity. This reagent reacts readily with 1-naphthol to form an osmiophilic quinoneimine group resulting in the transfer of electrons to cytochrome c which is subsequently oxidized by cytochrome oxidase. The quinoneimine group reacts with OSO4 to yield an electron dense pigment at the site of cytochrome oxidase activity.