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Abstract
Fungal electron transport systems (ETS) are branched, involving alternative NADH dehydrogenases and an alternative terminal oxidase. These alternative respiratory enzymes were reported to play a role in pathogenesis, production of antibiotics and excretion of organic acids. The activity of these alternative respiratory enzymes strongly depends on environmental conditions. Functional analysis of fungal ETS under highly standardised conditions for cultivation, sample processing and respirometric assay are still lacking. We developed a highly standardised protocol to explore in vivo the ETS—and in particular the alternative oxidase—in Penicillium ochrochloron. This included cultivation in glucose-limited chemostat (to achieve a defined and reproducible physiological state), direct transfer without any manipulation of a broth sample to the respirometer (to maintain the physiological state in the respirometer as close as possible to that in the chemostat), and high-resolution respirometry (small sample volume and high measuring accuracy). This protocol was aimed at avoiding any changes in the physiological phenotype due to the high phenotypic plasticity of filamentous fungi. A stable oxygen consumption (< 5% change in 20 minutes) was only possible with glucose limited chemostat mycelium and a direct transfer of a broth sample into the respirometer. Steady state respiration was 29% below its maximum respiratory capacity. Additionally to a rotenone-sensitive complex I and most probably a functioning complex III, the ETS of P. ochrochloron also contained a cyanide-sensitive terminal oxidase (complex IV). Activity of alternative oxidase was present constitutively. The degree of inhibition strongly depended on the sequence of inhibitor addition. This suggested, as postulated for plants, that the alternative terminal oxidase was in dynamic equilibrium with complex IV—independent of the rate of electron flux. This means that the onset of activity does not depend on a complete saturation or inhibition of the cytochrome pathway.
Highlights
The inner membrane of fungal mitochondria may contain—beside the standard components of the electron transport system (ETS)–several alternative redox enzymes (Fig 1): (i) up to three alternative external NAD(P)H dehydrogenases [1] or (ii) an alternative internal NAD(P)H dehydrogenase [2], both of which feed electrons to complex I into the quinone pool
Salicylhydroxamic acid (SHAM), benzhydroxamic acid (BHAM), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), 2,4-dinitrophenol (DNP), antimycin A and rotenone were purchased from Sigma-Aldrich and n-propyl gallate (n-PG) from Fluka
To achieve our goal, a rigorous standardisation was necessary on three levels: cultivation, sample preparation, and respirometric assay
Summary
The inner membrane of fungal mitochondria may contain—beside the standard components (complexes I, II, III, IV) of the electron transport system (ETS)–several alternative redox enzymes (Fig 1): (i) up to three alternative external NAD(P)H dehydrogenases [1] or (ii) an alternative internal NAD(P)H dehydrogenase [2], both of which feed electrons to complex I into the quinone pool. An (iii) alternative oxidase (AOX), works in parallel to the cytochrome c oxidase (COX) by reducing oxygen to water. These alternative redox enzymes have been suggested to be involved in many metabolic activities. The AOX is reported to be induced by a functionally compromised COX in Neurospora crassa [3, 4], or being involved in the pathogenesis of Aspergillus fumigatus [5] or active during the production of cephalosporin C by Acremonium chrysogenum [6]. The fungal complex III and the AOX have been targets for fungal pest management in agriculture [11]. The AOX has come into focus as a potential drug target of pathogenic fungi in humans [4, 5, 12]
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