A nanosecond time-resolved XFEL analysis of structural changes associated with CO release from cytochrome c oxidase.

Abstract

Bovine cytochrome c oxidase (CcO), a 420-kDa membrane protein, pumps protons using electrostatic repulsion between protons transferred through a water channel and net positive charges created by oxidation of heme <i>a</i> (Fe _<i>a</i> ) for reduction of O₂ at heme <i>a</i>₃ (Fe _<i>a</i>3). For this process to function properly, timing is essential: The channel must be closed after collection of the protons to be pumped and before Fe _<i>a</i> oxidation. If the channel were to remain open, spontaneous backflow of the collected protons would occur. For elucidation of the channel closure mechanism, the opening of the channel, which occurs upon release of CO from CcO, is investigated by newly developed time-resolved x-ray free-electron laser and infrared techniques with nanosecond time resolution. The opening process indicates that Cu_B senses completion of proton collection and binds O₂ before binding to Fe _<i>a</i>3 to close the water channel using a conformational relay system, which includes Cu_B, heme <i>a</i>₃, and a transmembrane helix, to block backflow of the collected protons.