NITRATE REDUCTASE IN CELL-FREE EXTRACTS OF A HAEMIN-REQUIRING STRAIN OF STAPHYLOCOCCUS AUREUS

Abstract

Nitrate-reductase activity was demonstrated in cell-free extracts of a mutant strain of S. aureus requiring either haemin or acetate plus purines and uracil for growth. Extracts from the mutant grown without haemin require either haemin or pyocy-anine for nitrate reduction in a system containing phosphate buffer, lactate, lactic dehydrogenase, cysteine and nicotinamide-adenine-dinucleotide (NAD). Flavin-mononucleotide (FMN) or flavin-adenine-dinucleotide (FAD) stimulate the reduction in the presence of haemin but not with pyocyanine. Extracts from the parent organism, which does not require haemin for growth, or from the mutant grown with haemin reduce nitrate in the absence of haemin or pyocyanine, though the latter is stimulatory. Haemin-dependent nitrate reductase is inhibited by 2-n-heptyl-4-hydroxyquinoline N-oxide. The inhibitory effects of metal chelators cyanide and azide and the stimulation by Fe suggest the participation of this metal in nitrate reduction. Nitrate reductase is induced by growth in the presence of nitrate and is repressed by aeration. The repression occurs irrespective of the presence of haemin in the medium. Nitrate reductase is present in the soluble and particulate portions of the mutant organism. The haemin-dependent activity is located in the particles. In organisms grown without haemin there is more soluble enzyme than in organisms grown with this factor. Particles from the mutant strain, grown without haemin, form a haemoprotein when incubated with haemin and cysteine; the reduced absorption maxima are similar to those of cytochrome-b1 and to those in particles from the parent organism. The S. aureus mutant forms the protein component of cytochrome-b1 when grown without haemin; this combines with haemin to give cytochrome-b1, which participates in electron transfer to nitrate, mediated by nitrate reductase.