Pulse Radiolysis Studies of Temperature Dependent Electron Transfers among Redox Centers in ba3-Cytochrome c Oxidase from Thermus thermophilus: Comparison of A- and B-Type Enzymes

The functioning of cytochrome c oxidases involves orchestration of long-range electron transfer (ET) events among the four redox active metal centers. We report the temperature dependence of electron transfer from the CuAr site to the low-spin heme-(a)bo site, i.e., CuAr + heme-a(b)o-* CuAo+ heme-a(b)r in three structurally characterized enzymes: A-type aa3 from Para coccus denitrificans (PDB code 3HB3) and bovine heart tissue (PDB code 2ZXW), and the B-type ba3 from T. thermophilus (PDB codes 1EHK and 1XME). k,T data sets were obtained with the use of pulse radiolysis as described previously. Semiclassical Marcus theory revealed that lambda varies from 0.74 to 1.1 eV, Hab, varies from similar to 2 x 10-5 eV (0.16 cm-1) to similar to 24 x 10-5 eV (1.9 cm-1), and beta D varies from 9.3 to 13.9. These parameters are consistent with diabatic electron tunneling. The II-Asp111Asn CuA mutation in cytochrome ba3 had no effect on the rate of this reaction whereas the II-Met160Leu CuA-mutation was slower by an amount corresponding to a decreased driving force of similar to 0.06 eV. The structures support the presence of a common, electron-conducting "wire" between CuA and heme-a(b). The transfer of an electron from the low-spin heme to the high-spin heme, i.e., heme-a(b)r + heme-a3o-* heme-a(b)o + heme-a3r, was not observed with the A-type enzymes in our experiments but was observed with the Thermus ba3; its Marcus parameters are lambda = 1.5 eV, Hab = 26.6 x 10-5 eV (2.14 cm-1), and beta D = 9.35, consistent also with diabatic electron tunneling between the two hemes. The II-Glu15Ala mutation of the K-channel structure, similar to 24 angstrom between its CA and Fe -a3, was found to completely block heme-br to heme-a3o electron transfer. A structural mechanism is suggested to explain these observations.