ИСТИНА

Cytochrome bd is expressed under low oxygen tension and has high affinity for O2. The enzyme as isolated is a mixture of two 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 20–50%. Both forms can be reversibly destroyed either by oxidation (anaerobically or aerobically) or by depletion of oxygen at Eh where the enzyme remains in one-electronreduced state. Binding of O2 to heme d was studied using the quasi-equilibrium OTTLE spectroelectrochemistry, where oxygen was allowed to equilibrate with the enzyme at a given Eh. Under the anaerobic conditions heme d has Em app ~+260 mV (vs.NHE, pH 7, 0.1% SML); at 1.2 mM O2, the Em app value becomes +495 mV. The [O2] -dependence of Em app is linear at the concentrations above ~10 mM with the slope -60 mV/pO2 and the effective dissociation constant KD ~ 150 nM O2. The latter value differs from the earlier data (KD ~ 280 nM) where the oxygen affinity was directly measured in one-electron-reduced isolated enzyme. The difference is attributed to the fact that in the presence of O2, the enzyme catalyzes a steady-state flux of electrons supplied by the working electrode. Modeling proves that in the steady-state, the KD value of 280 nM is reached when the kinetics of the O -> A and A -> (F) -> O transitions are the same and limited by the electron delivery from the working electrode, and the A -> F transition is much faster. The O -> A and A -> F transitions show similar Em app values over the broad [O2] range. We propose that in the state F, heme d has an unusually low redox potential comparable to that of the state A enzyme.