A time-resolved resonance Raman analysis shows that the vinyl stretching band (1629 cm(-1)) of the O-2-binding heme of cytochrome c oxidase shifts to 1627 cm(-1) instantaneously upon photolysis of CO and remains for at least 5 ms before reaching the static band (1626 cm(-1)). Within the same time scale, an intermediate vinyl bending mode of another heme appears at 435 cm(-1). These results suggest that both hemes cooperatively control O-2 binding by forming an intermediate conformation for effective proton pumping.