Role of copper ion in regulating ligand binding in a myoglobin-based cytochrome C oxidase model.

Cytochrome c oxidase (CcO), the terminal enzyme in the mitochondrial respiratory chain, catalyzes the four-electron reduction of dioxygen to water in a binuclear center comprised of a high-spin heme (heme a(3)) and a copper atom (Cu(B)) coordinated by three histidine residues. As a minimum model for CcO, a mutant of sperm whale myoglobin, named Cu(B)Mb, has been engineered, in which a copper atom is held in the distal heme pocket by the native E7 histidine and two nonnative histidine residues. In this work, the role of the copper in regulating ligand binding in Cu(B)Mb was investigated. Resonance Raman studies show that the presence of copper in CO-bound Cu(B)Mb leads to a CcO-like distal heme pocket. Stopped-flow data show that, upon the initiation of the CO binding reaction, the ligand first binds to the Cu(+); it subsequently transfers from Cu(+) to Fe(2+) in an intramolecular process, similar to that reported for CcO. The high CO affinity toward Cu(+) and the slow intramolecular CO transfer rate between Cu(+) and Fe(2+) in the Cu(B)Mb/Cu(+) complex are analogous to those in Thermus thermophilus CcO (TtCcO) but distinct from those in bovine CcO (bCcO). Additional kinetic studies show that, upon photolysis of the NO-bound Cu(B)Mb/Cu(+) complex, the photolyzed ligand transiently binds to Cu(+) and subsequently rebinds to Fe(2+), accounting for the 100% geminate recombination yield, similar to that found in TtCcO. The data demonstrate that the Cu(B)Mb/Cu(+) complex reproduces essential structural and kinetic features of CcO and that the complex is more akin to TtCcO than to bCcO.