Single-O ₂ ligation of hemoglobin links aerobic and anaerobic metabolism

Oxygen (O ₂ ) binding and release by hemoglobin (Hb) are governed by cooperative interactions among its four subunits. During incremental workload exercise, femoral venous oxyhemoglobin (O ₂ Hb) saturation exhibits a reproducible, momentary increase at the gas exchange threshold—coinciding with the inflection point of the in vivo O ₂ non-equilibrium curve (ONC). This suggests a transient shift in Hb’s binding dynamics. We hypothesized that at this threshold, Hb tetramers carrying ≤1 bound O ₂ become predominant. In this state, the last bound O ₂ promotes further cooperative binding, but its release confers no cooperative advantage for unloading, biasing toward O ₂ rebinding. Using the O ₂ equilibrium curve models of Dash et al. (2016) and Adair, we computed the distribution of Hb’s O ₂ 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 O ₂ indeed dominated. This ligation-state distribution is consistent with Perrella et al.’s (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 O ₂ transport physiology, framing the Hb-Bohr system as a proportional-integral controller of tissue oxygenation.