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Simplified isolation and molecular composition of NADH dehydrogenase of the respiratory chain.

ABSTRACT: A simplified procedure for the isolation of NADH dehydrogenase from the inner membrane of ox heart mitochondria is presented which permits relatively rapid preparation of the enzyme in a more stable form than that afforded by published methods. The protein thus isolated displays more than eight different subunits in gel electrophoresis under denaturing conditions, three of which are also present in the "low-molecular-weight form' of the enzyme prepared under more drastic conditions. Complex I contains several subunits, mostly of low molecular weight, not seen in soluble purified NADH dehydrogenase. It is suggested that some of these may be 'binding peptides' necessary in linking NADH dehydrogenase to ubiquinone reduction, analogously to the role of small peptides in linking succinate dehydrogenase to ubiquinone. The dehydrogenase isolated by the rapid method contains equimolar amounts of non-haem iron and labile sulphur, but on further manipulation non-haem iron (but no labile sulphur) is lost, resulting in ratios of S/Fe in excess of unity, as previously reported for preparations isolated by longer procedures.

SUBMITTER: Paech C

PROVIDER: S-EPMC1158253 | biostudies-other | 1982 May

REPOSITORIES: biostudies-other

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Project description:The thermotolerant yeast Ogataea parapolymorpha (formerly Hansenula polymorpha) is an industrially relevant production host that exhibits a fully respiratory sugar metabolism in aerobic batch cultures. NADH-derived electrons can enter its mitochondrial respiratory chain either via a proton-translocating complex I NADH-dehydrogenase or via three putative alternative NADH dehydrogenases. This respiratory entry point affects the amount of ATP produced per NADH/O2 consumed and therefore impacts the maximum yield of biomass and/or cellular products from a given amount of substrate. To investigate the physiological importance of complex I, a wild-type O. parapolymorpha strain and a congenic complex I-deficient mutant were grown on glucose in aerobic batch, chemostat, and retentostat cultures in bioreactors. In batch cultures, the two strains exhibited a fully respiratory metabolism and showed the same growth rates and biomass yields, indicating that, under these conditions, the contribution of NADH oxidation via complex I was negligible. Both strains also exhibited a respiratory metabolism in glucose-limited chemostat cultures, but the complex I-deficient mutant showed considerably reduced biomass yields on substrate and oxygen, consistent with a lower efficiency of respiratory energy coupling. In glucose-limited retentostat cultures at specific growth rates down to ∼0.001 h-1, both O. parapolymorpha strains showed high viability. Maintenance energy requirements at these extremely low growth rates were approximately 3-fold lower than estimated from faster-growing chemostat cultures, indicating a stringent-response-like behavior. Quantitative transcriptome and proteome analyses indicated condition-dependent expression patterns of complex I subunits and of alternative NADH dehydrogenases that were consistent with physiological observations.IMPORTANCE Since popular microbial cell factories have typically not been selected for efficient respiratory energy coupling, their ATP yields from sugar catabolism are often suboptimal. In aerobic industrial processes, suboptimal energy coupling results in reduced product yields on sugar, increased process costs for oxygen transfer, and volumetric productivity limitations due to limitations in gas transfer and cooling. This study provides insights into the contribution of mechanisms of respiratory energy coupling in the yeast cell factory Ogataea parapolymorpha under different growth conditions and provides a basis for rational improvement of energy coupling in yeast cell factories. Analysis of energy metabolism of O. parapolymorpha at extremely low specific growth rates indicated that this yeast reduces its energy requirements for cellular maintenance under extreme energy limitation. Exploration of the mechanisms for this increased energetic efficiency may contribute to an optimization of the performance of industrial processes with slow-growing eukaryotic cell factories.

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Simplified isolation and molecular composition of NADH dehydrogenase of the respiratory chain.

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