In vivo quantification of mitochondrial O ₂ affinity in human skeletal muscle using ¹ H‐magnetic resonance spectroscopy of deoxy‐myoglobin

Mitochondrial oxygen (O 2 ) 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 O 2 affinity (i.e. P 50 ) of the skeletal muscle, we used proton‐magnetic resonance spectroscopy ( 1 H‐MRS) of deoxymyoglobin (dMb) during a circulatory occlusion of the lower limb to simultaneously quantify intracellular partial pressure of O 2 (i ) and resting tissue‐specific O 2 consumption () in the gastrocnemius muscle of sedentary young adults. Under these resting conditions, the –i relationship conformed to a Michaelis–Menten kinetics model (goodness of fit: r 2 = 0.84 ± 0.13). The estimated V ss , reflecting basal metabolic rate, reached 0.20 ± 0.06 mM min −1 , and the apparent mitochondrial P 50 was 0.50 ± 0.38 mmHg. V ss and P 50 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 O 2 affinity ( P 50 ≈ 0.5 mmHg). In addition, it quantitatively identifies the range across which oxidative phosphorylation in the skeletal muscle is O 2 ‐independent (resting i under physiological conditions) or becomes O 2 ‐sensitive (severe exercise and hypoxia). Moreover, the finding that muscle respiratory flux was associated with the apparent mitochondrial O 2 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. image Key points Mitochondrial oxygen affinity ( P 50 ) is central to oxidative phosphorylation, yet has not been directly quantified in human skeletal muscle in vivo . Using deoxymyoglobin 1 H‐MRS during 8 min of lower‐limb ischaemia, we simultaneously measure intracellular partial pressure of O 2 (i ) and resting tissue‐specific O 2 consumption () in the resting gastrocnemius of healthy young adults. The – relationship followed Michaelis–Menten kinetics, yielding a V ss of 0.20 mM min −1 and an apparent mitochondrial P 50 of 0.50 mmHg, indicating very high O 2 affinity. Resting apparent mitochondrial P 50 was far below previously reported i (∼34 mmHg), indicating a wide O 2 ‐independent range and identifying conditions (severe hypoxia, high‐intensity exercise) in which oxidative phosphorylation becomes O 2 ‐sensitive. V ss and P 50 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.