Studies of respiratory components and oxidative phosphorylation in mitochondria of mi-1 Neurospora crassa

Abstract

Oxidative phosphorylation has been demonstrated with mitochondria of the mi-1 respiratory mutant of Neurospora crassa. The P/O ratios observed with these mitochondria were approximately 0.8 with citrate and 0.4 with either externally added reduced nicotinamide adenine dinucleotide (NADH), succinate, or ascorbate-tetramethyl-p-phenylenediamine (TPD). These P/O ratios suggest that there are only two sites of phosphorylation in mitochondria isolated from young (20 to 24 h) cultures of the mi-1 mutant. The energy-dependent reduction of NAD(+) with succinate and the phosphorylation associated with ascorbate-TPD oxidation indicate that the first and the third sites of energy coupling are present in this mutant. Difference spectra of mitochondria from young cultures of the mi-1 mutant revealed the presence of cytochrome c. Cytochromes b and a + a(3) were not detected. However, in the presence of antimycin A, a small peak in the Soret region at 430 nm was observed. A carbon monoxide difference spectrum revealed the presence of a component of the respiratory chain with a spectrum similar to that of cytochrome o. It is of interest that respiratory inhibitors such as antimycin A, 2-n-nonylhydroxyquinoline N-oxide, and cyanide abolished phosphorylation but only partially inhibited oxidation. It is postulated that the mi-1 respiratory system contains two pathways of electron transport-the first is associated with a phosphorylating pathway, whereas the second is a non-phosphorylating electron transport pathway.