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:::: Updated terminology: 2018-07-04
The classical question for oxidative phosporylation (OXPHOS) ‘What does it do?’ (1) is extended in mitochondrial physiology by asking How fast can it go? Accordingly, approaches of bioenergetics are extended for measuring the upper limits of OXPHOS and Electron transfer-pathway (ET-pathway) capacity. Estimates of maximum mitochondrial performance (2) provide essential reference points for analysis of metabolic control mechanisms and diagnosis of mitochondrial function in health and disease.
Why do most studies over the past 55 years not report upper limits of OXPHOS, and how are these determined (3)? OXPHOS capacity is measured in mitochondrial preparations, stimulated by saturating [ADP] and [P<sub>i</sub>], and supported by NADH-linked substrates (N) or succinate (S) to determine site-specific P:O ratios (4). This limits flux by selective electron gating (Fig. 1). N-input (e.g. pyruvate&malate) yields 0.5-0.8, succinate 0.7-0.9 of combined NS-OXPHOS capacity, in skeletal muscle mitochondria of mammals and birds. Consistent with the convergent structure of the ET-pathway (5) (Fig. 1), NS substrate combinations boost maximum mitochondrial performance (2,3,5,6).
Why is it important to determine the upper limit of OXPHOS capacity?
(1) At the integrated organismic level, ergometric VO<sub>2<sub>max</sub></sub> depends on matched capacities of elements in the respiratory cascade, from lung to mitochondria (7). Upscaled mitochondrial capacities entail revisions of established models of control.
(2) Discrepancies of low OXPHOS capacity in isolated mitochondria (imt) versus intact cells (8) are explained by (i) electron gating in mt-preparations. In permeabilized human fibroblasts, N- or S-substrate supply supported only 77% and 68%, respectively, of combined NS-electron input (9). (ii) ET-pathway but not OXPHOS capacity can be determined in intact cells. The OXPHOS/ET-pathway flux ratio (NS) was 0.5 in permeabilized cells. Taken together, conventional OXPHOS-capacity is <40% of ET-pathway(NS) capacity in permeabilized fibroblasts, and full agreement was obtained of ET-pathway capacities in permeabilized and intact cells. In these cells, mild uncoupling does not reduce the apparent reserve capacity for phosphorylation, since OXPHOS is not limited by ET-pathway but by the phosphorylation system.
(3) Discrepancies are resolved between apparently high excess capacities of cytochrome ''c'' oxidase (CIV) in imt (in protocols with electron gating) versus permeabilized muscle fibres (measured with combined NS-electron input, without explanation (10)). The apparent difference is not a property of the type of mitochondrial preparation. In general, thresholds and spare capacities of OXPHOS components downstream of Q are overestimated without reference to the upper limit of mitochondrial performance, impacting any functional evaluation of pathological enzymatic defects.
(4) On the integrated pathway level, electron gating in mt-preparations causes underestimation of flux control coefficients for enzyme steps downstream of Q.
(5) Substrate kinetics (ADP, P<sub>i</sub>, O<sub>2</sub>) yields ''K''<sub>m</sub>’ values, which need re-assessment at high NS-supported flux, since the ''K''<sub>m</sub>’ is a function of enzyme turnover (11).
(6) Besides N and S, ETF and glycerophosphate dehydrogenase are gates for electron transfer converging at the Q-junction. Corresponding additive or competitive effects on respiration need to be re-investigated on the basis of NS-electron input.
(7) Mitochondrial density is the primary determinant of the upper limit of respiratory performance of a cell, requiring the measurement of mitochondrial quantity. In addition, mitochondrial quality differs between cell types, tissues, and species. Since substrate control varies, comparative mitochondrial physiology is functionally meaningful only if it is based on the upper limit of mitochondrial respiratory performance.
Enhancing but less than completely additive effects are observed with NS-electron input. These combination effects are interpreted as synergistic or antagonistic, depending on the model of additivity (12). A new mathematical definition of additivity resolves these ambiguities by introducing incomplete additivity intermediate between antagonistic suppression below zero additivity, and synergistic activation above complete additivity. This conceptual framework, based on the growing evidence of incompletely additive effects of convergent electron transfer into the Q-junction (2,13) may be instrumental to catalyze an overdue paradigm change from bioenergetics to mitochondrial physiology, to establish appropriate standards for evaluation of mitochondrial respiratory control at the upper limits of aerobic performance and at the limiting threshold levels of disease.
Contribution to Mitofood COST Action FAO602.
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