In vivo quantification of mitochondrial O₂ affinity in human skeletal muscle using ¹H-magnetic resonance spectroscopy...

Mitochondrial oxygen (O₂) affinity is a fundamental determinant of oxidative phosphorylation capacity, which has yet to be directly measured in human skeletal muscle in vivo. To determine the apparent mitochondrial O₂ affinity (i.e. P₅₀) of the skeletal muscle, we used proton-magnetic resonance spectroscopy (¹H-MRS) of deoxymyoglobin (dMb) during a circulatory occlusion of the lower limb to simultaneously quantify intracellular partial pressure of O₂ (i $P_{O_2}$ ) and resting tissue-specific O₂ consumption ( ${\dot{V}}_{O_2}$ ) in the gastrocnemius muscle of sedentary young adults. Under these resting conditions, the ${\dot{V}}_{O_2}$ -i $P_{O_2}$ relationship conformed to a Michaelis-Menten kinetics model (goodness of fit: r² = 0.84 ± 0.13). The estimated V_ss, reflecting basal metabolic rate, reached 0.20 ± 0.06 mM min^-1, and the apparent mitochondrial P₅₀ was 0.50 ± 0.38 mmHg. V_ss and P₅₀ were positively correlated (r = 0.85, P = 0.0009). This strong correlation remained after log transformation (r = 0.82, P = 0.0020). These results, obtained in human muscles in vivo, demonstrate that mitochondrial respiration exhibits a very high O₂ affinity (P₅₀ ≈ 0.5 mmHg). In addition, it quantitatively identifies the range across which oxidative phosphorylation in the skeletal muscle is O₂-independent (resting i $P_{O_2}$ under physiological conditions) or becomes O₂-sensitive (severe exercise and hypoxia). Moreover, the finding that muscle respiratory flux was associated with the apparent mitochondrial O₂ affinity suggests additional regulatory mechanisms within the respiratory chain to fine-tune oxidative phosphorylation to muscle ATP demand even at rest. This study provides a robust quantitative framework for interpreting in vivo respiratory control in both health and disease. KEY POINTS: Mitochondrial oxygen affinity (P₅₀) is central to oxidative phosphorylation, yet has not been directly quantified in human skeletal muscle in vivo. Using deoxymyoglobin ¹H-MRS during 8 min of lower-limb ischaemia, we simultaneously measure intracellular partial pressure of O₂ (i $P_{O_2}$ ) and resting tissue-specific O₂ consumption ( ${\dot{V}}_{O_2}$ ) in the resting gastrocnemius of healthy young adults. The ${\dot{V}}_{O_2}$ - $P_{O_2}$ relationship followed Michaelis-Menten kinetics, yielding a V_ss of 0.20 mM min^-1 and an apparent mitochondrial P₅₀ of 0.50 mmHg, indicating very high O₂ affinity. Resting apparent mitochondrial P₅₀ was far below previously reported i $P_{O_2}$ (∼34 mmHg), indicating a wide O₂-independent range and identifying conditions (severe hypoxia, high-intensity exercise) in which oxidative phosphorylation becomes O₂-sensitive. V_ss and P₅₀ were positively correlated (r = 0.85), suggesting in vivo coupling between respiratory flux and oxygen affinity and providing a potential quantitative framework for interpreting mitochondrial respiratory control in health and disease.