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Cytochrome bd is typically expressed under low oxygen tension and has high affinity for O2. The enzyme as isolated is a mixture of two stable or metastable forms: (i) ferrous heme d bound to molecular oxygen (state “A”), and (ii) ferryl oxene heme d (state “F”), with the latter form contribution of ca. 20-50%. Both forms can be fully but reversibly destroyed by (a) anaerobic (at Eh > +380 mV vs. NHE, pH 7, 0.1% SML) or aerobic (> +620 mV) oxidation or (b) depletion of oxygen at redox potentials where the enzyme mostly remains in one-electron-reduced state; in both cases state “O” is formed. Binding of O2 to heme d as a function of Eh was studied using the quasi-equilibrium OTTLE spectroelectrochemistry, where oxygen at varying concentrations was allowed to equilibrate with the enzyme at a given redox potential. Under the anaerobic conditions heme d has the apparent midpoint potential Emapp ~ +260 mV with both hemes b mostly oxidized (1); at 1.2 mM O2, the potential value becomes +495+-5 mV. The [O2]-dependence of Emapp is essentially linear at the concentrations above 10 mM with the slope -60 mV/pO2 and the effective dissociation constant for the reduced heme d, KD(eff) ~ 150 +-20 nM O2. The latter value differs from the earlier data on the heme d oxygen affinity directly measured in one-electron-reduced isolated enzyme yielding KD(direct) ~ 280 nM. The difference is attributed to the fact that in the presence of oxygen, the enzyme slowly turns over catalyzing a steady-state flux of electrons across the electrochemical cell. The turnover numbers of the enzyme at each redox potential can be determined in situ from the values of the cell current (IWE) taking into account the unspecific current in the absence of the enzyme. Kinetic modeling proves that in the steady-state, the KD(eff) of 280 nM can be reached when the rate constants for the O -> A and O -> A transitions are equal, which is indeed true, provided that both constants are limited by the interaction of the enzyme with a reduced redox mediator, and when the (mediator-independent) A -> F transition is much faster. The O -> A and A -> F transitions show similar Emapp values during the redox titration over the broad [O2] range. We propose that the state F of heme d in cytochrome bd has an unusually low redox potential, which value is comparable to that of heme d in the A state.