Nanosecond Time-Resolved Infrared Basis for a Bulge of the Transmembrane Helix Between Hemes A and A3 to Facilitate Highly Efficient Proton Pumping by Bovine Heart Cytochrome C Oxidase

Recent X-ray structural analyses show that net positive charges on a heme iron site (heme a) of bovine cytochrome c oxidase, created upon electron donation to the O2-reduction site, electrostatically drives proton-pump through a hydrogen-bond network to the positive side of mitochondrial inner membrane [1, 2]. The four electron equivalents for complete reduction of O2 at the fully reduced O2-reduction site are transferred one at a time, each, coupled with pumping of one proton equivalent, giving four intermediate species, F, O, E and R (from P). X-ray structures of P, F, O and R suggested that the water channel which connects the negative side space with the hydrogen bond network is kept closed by Ser382 bulge of the trans-membrane-helix during the transfer of four electron equivalents for complete reduction of the bound O2. The closure blocks effectively proton back-leakage from the hydrogen-bond network.The structural basis for complete protonation of the hydrogen-bond network before the O2-binding prerequisite for the efficient energy transduction was explored using a newly developed nanosecond time-resolved infrared apparatus for aqueous protein system. A transient CO-binding to CuB, after Fea3-CO photolysis, was discovered to open the water-channel by eliminating the Ser382 bulge. The infrared and X-ray structural results suggest that, sensing protonation state of the hydrogen-bond network, a relay system including CuB, O2, Fea3 and two a-helix turns extending to Ser382 facilitates effective proton collection and timely water-channel closure by conformational changes in the Ser382-containing segment, thereby ensuring efficient energy transduction.[1] Muramoto K., et al (2010) Proc. Natl. Acad. Sci. USA 107: 7740-7745.[2] Yoshikawa S., et al (2011) Ann. Rev. Biophys. 40: 205-223.