Comparison of the respiratory chain of Neurospora crassa wild type and the mi-mutants mi-1 and mi-3.

Abstract

Mitochondria from maternally inherited mutants of Neurospora crassa mi‐1 (poky) and mi‐3 were examined with respect to the components of the respiratory chain, their properties and arrangement. Furthermore the sites of oxidative phosphorylation were localized. An evaluation procedure which permits the quantitative determination of the various b‐type and c‐type cytochromes is given. All mutant mitochondria contain the same cytochromes as wild type mitochondria, such as cytochrome a,a3, cytochrome c and c1 and cytochrome b562 and b556 (the indices refer to the maxima at low temperature). Yet the molar ratios of the cytochromes; a:c:c1:b562:b556 differ widely: in the wild type the ratios are 1.0:2.9:1.0:0.9:0,9; in mi‐1 the ratios are <0.05:3.6:1.0:0.05:0.3; and in mi‐3 the ratios are 0.3:3.2:1.0:0.9:0.8. The content (μmol/g protein) of the reference cytochrome c1 varies slightly from 0.34 in, the wild type to 0.39 in mi‐1 and 0.32 in mi‐3. The main feature of the cytochrome pattern in mi‐1 is a very low content of cytochrome a,a3 and b562; besides that the content of cytochrome c is increased by one third of the normal value. In mi‐3 only the content of cytochrome a,a3is diminished. The comparatively low content of ubiquinone in the wild type is increased 8‐fold in mi‐1 and 5‐fold in mi‐3. The content of mitochondria1 NAD remains largely unchanged. Both mitochondria from mi‐1 and mi‐3 show antimycin‐ and cyanide‐insensitive respiration. The share of uninhibited respiration depends on the activity of the different dehydrogenases and on the ratio of the capacities of both oxidase pathways. The insensitive respiration decreases with the age of the hyphae culture. The ADP/O ratio in mi‐I mitochondria is lowered, due to the cyanide‐insensitive oxidase pathway. This pathway is found to include one phosphorylation step with substrates linked to endogenous NADH. Exogenous NADH and NADPH are oxidized similarly to succinate, involving two phosphorylation sites on the cyanide‐sensitive path but no phosphorylation site on the insensitive path. Inhibitors distinguish the two oxidase pathways: the normal pathway is sensitive to cyanide and antimycin, the second pathway to salicylhydroxamate. NAD‐linked and ubiquinone‐linked substrates are oxidized by both pathways whereas tetramethyl‐p‐phenylenediamine plus ascorbate is oxidized only by the normal cyanide‐sensitive pathway. The redox state of ubiquinone in relation to antimycin and salicylhydroxamate indicates participation of ubiquinone in both paths of the mi‐1 mutant. In wild type, cytochrome b562 is largely oxidized in anaerobic uncoupled state but can be reduced to about 60–70% by ATP, whereas cytochrome b556 is largely reduced under both conditions. Cytochrome b562 is completely reduced in the antimycin‐inhibited state and becomes oxidized on reduction of cytochrome cl. In analogy to mammalian mitochondria cytochrome b562 is defined as cytochrorne bT and cytochrome b556 as cytochrome bK. In contrast to the wild type, cytochrome b556 in mi‐1 remains largely oxidized even with antimycin present. Titration with antimycin gives nonlinear curves with a titer of complete inhibition at about equimolar ratio of antimycin to cytochrome bT. In the mi‐1 mutant the antimycin titer strongly decreases parallel to the loss of cytochrome bT.