Redox conditions and protein oxidation in plant mitochondria

Redox conditions and protein oxidation in plant mitochondria NAD(P)H has a central position in respiratory metabolism. It is produced by a large number of enzymes, e.g. the Krebs cycle dehydrogenases, in the mitochondrial matrix and is oxidised by, amongst others, the respiratory chain. Most of this NAD(P)H appears to be bound to proteins, in fact free NAD(P)H – an important parameter in metabolic regulation - has never been observed in mitochondria. We have estimated free and bound NAD(P)H in isolated plant mitochondria under different metabolic conditions. The fluorescence spectra of free and bound NADH was determined and used to deconvolute fluorescence spectra of actively respiring mitochondria. Most of the mitochondrial NADH is bound in states 2 and 4. The amount of free NADH is lower but relatively constant even increasing a little in state 3 where it is about equal to bound NADH. The consequences for the regulation of respiratory metabolism will be considered. When the conditions in the mitochondria are particularly reducing such as in state 4 or in the presence of inhibitors of the respiratory chain the production of Reactive Oxygen Species is increased (Ref. 1). This can lead to damage to proteins, lipids and DNA. We are studying the oxidation of proteins. The formation of N-formylkynurenin by dioxygenation of tryptophan was detected in peptides from rice leaf and potato tuber mitochondria by mass spectrometry. Oxidized tryptophan was detected in 27 peptides representing 17 different proteins. With one exception, the oxidation-sensitive aconitase, all of these proteins were either redox active themselves, e.g., aldehyde dehydrogenase and superoxide dismutase, or subunits in redox-active enzyme complexes, such as glycine decarboxylase and the respiratory complexes I, III and IV. The same site was modified in (i) several adjacent spots containing the P-protein of the glycine decarboxylase complex, (ii) two different isoforms of the mitochondrial processing protease in complex III and (iii) superoxide dismutase in both rice and potato mitochondria. This indicates that tryptophan oxidation is a selective process. 1) Møller, I.M. 2001. Plant mitochondria and oxidative stress. Electron transport, NADPH turnover and metabolism of reactive oxygen species. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 561-591