Evidence for the presence of two conformations of the heme a 3-CuB pocket of cytochrome caa 3 from Thermus thermophilus

Abstract

Resonance Raman (RR) and "light" minus "dark" Fourier transform infrared (FTIR) difference spectra are reported for the CO-bound caa3 oxidase from Thermus thermophilus. Two Fe-CO stretching modes at 518 and 507 cm-1, the Fe-C-O bending mode at 570 cm-1, and three C-O modes of heme a3 at 1958, 1967, and 1973 cm -1 have been identified in the RR and FTIR spectra, respectively. The FTIR "light" minus "dark" spectrum indicates the formation of CuBCO as revealed by its ν(CO) at 2060/2065 cm-1. We assign the bands at 518 (νFe-CO) and 1967/1973 cm-1 (νC-O) as the α-conformation. We also assign the bands at 507 and 1958 cm-1 (νC-O) as originating from the β-conformation of the enzyme. A frequency upshift of the heme a3 Fe-His mode is observed subsequent to CO photolysis from 209 cm-1 in the equilibrium deoxy enzyme to 214 cm-1 in the photoproduct. The caa3 data, distinctly different from those of ba3 oxidase, are discussed in terms of the coupling of the α- and β-conformations that occur in heme-copper oxidases with catalytic function. The dynamics between the heme a3 and heme a propionates as revealed by the perturbation of the bending vibrations δprop of hemes a and a3 at 385 and 392 cm-1, respectively, induced upon CO binding to heme a3 is discussed in terms of the protonic connectivity between the heme a ring-D propionate/Arg site with that of the heme a 3 ring-D propionate-H2O site that leads to the highly conserved in the heme-copper oxidases water pool. © 2011 American Chemical Society.