Oxidative Phosphorylation in Neurospora Mitochondria

Abstract

Abstract Oxidative phosphorylation has been studied in mitochondria from wild type and poky strains of Neurospora crassa and from wild type that had acquired the poky phenotype as a result of growth in the presence of chloramphenicol. Wild type mitochondria were found to be tightly coupled, with respiratory control ratios as high as 5.8 and P:O ratios as high as 1.9. Results with a variety of substrates and inhibitors led to the conclusion that most of the oxidative phosphorylation takes place at Sites 2 and 3. Site 1 is quite inefficient, judged by the small difference in phosphorylation efficiency between NAD+-linked substrates and succinate and also by the relatively low level of reduction of endogenous NAD+ during respiration with succinate (+ATP) (27 ± 4% in Neurospora compared with as much as 75% in animal mitochondria). Poky and chloramphenicol-induced mitochondria show little respiratory control and low P:O ratios under most experimental conditions. In these mitochondria the cytochrome system is known to be impaired, and a large part of the respiration is mediated by an alternate oxidase that is resistant to cyanide and antimycin. Essentially all of the oxidative phosphorylation is inhibited by cyanide or antimycin, however, suggesting that it is associated with the residual cytochrome system rather than with the alternate oxidase. Site 1 seems to be absent altogether in poky mitochondria; Sites 2 and 3 are both present but at reduced efficiency. It can be calculated that the over-all electron flux in poky is partitioned so that the cytochrome system operates at maximal activity while the nonphosphorylating alternate oxidase is used only to accommodate surplus electron flux.