Single-O₂ ligation of hemoglobin links aerobic and anaerobic metabolism

Oxygen (O₂) binding and release by hemoglobin (Hb) are governed by cooperative interactions among its four subunits. During incremental workload exercise, femoral venous oxyhemoglobin (O₂Hb) saturation exhibits a reproducible, momentary increase at the gas exchange threshold-coinciding with the inflection point of the in vivo O₂ nonequilibrium curve (ONC). This suggests a transient shift in Hb's binding dynamics. We hypothesized that at this threshold, Hb tetramers carrying ≤1 bound O₂ become predominant. In this state, the last bound O₂ promotes further cooperative binding, but its release confers no cooperative advantage for unloading, biasing toward O₂ rebinding. Using the O₂ equilibrium curve models of Dash et al. (Eur J Appl Physiol 116: 97-113, 2016) and Adair, we computed the distribution of Hb's O₂ ligation states across 12 pooled mean femoral venous blood samples from incremental workload cardiopulmonary exercise testing of five healthy male participants. At the gas exchange threshold-where the ONC inflects and flattens-tetramers with ≤1 O₂ indeed dominated. This ligation-state distribution is consistent with Perrella et al.'s (J Biol Chem 274: 2605-2608, 1999) cryogenic resolution of native human Hb, which shows that carbon monoxide-ligated Hb tetramers peak at ∼15%-20% saturation, matching femoral venous ranges at the gas exchange threshold. Our results suggest that, at sufficiently low O₂Hb saturation, Hb may favor O₂ rebinding over cooperative unloading. We propose that glycolytic proton production and other Bohr effectors may counter this predicted binding bias supporting continued O₂ unloading. If confirmed, this mechanism unifies long-standing controversies in O₂ transport physiology, framing the Hb-Bohr system as a proportional-integral controller of tissue oxygenation.NEW & NOTEWORTHY Anaerobic metabolism is usually viewed as a fallback when oxygen delivery becomes insufficient. Our analysis suggests a different role: it may preserve oxygen delivery by correcting a hemoglobin-binding bias that emerges at low oxyhemoglobin saturation. When hemoglobin tetramers carry one or no oxygen molecules, the remaining bound oxygen can promote cooperative rebinding but not cooperative release. Glycolytic proton production and other Bohr effectors may counter this bias, restoring oxygen release linking lactate, hypoxia, and cooperativity.