title: Observation of the equilibrium CuB-CO complex and functional implications of the transient heme a3 propionates in cytochrome ba3-CO from Thermus thermophilus. Fourier transform infrared (FTIR) and time-resolved step-scan FTIR studies.
abstract: We report the first evidence for the existence of the equilibrium Cu(B)1+-CO species of CO-bound reduced cytochrome ba(3) from Thermus thermophilus at room temperature. The frequency of the C-O stretching mode of Cu(B)1+-CO is located at 2053 cm(-1) and remains unchanged in H(2)O/D(2)O exchanges and, between pD 5.5 and 9.7, indicating that the chemical environment does not alter the protonation state of the Cu(B) histidine ligands. The data and conclusions reported here are in contrast to the changes in protonation state of Cu(B)-His-290, reported recently (Das, T. K., Tomson, F. K., Gennis, R. B., Gordon, M., and Rousseau, D. L. (2001) Biophys. J. 80, 2039-2045 and Das, T. P., Gomes, C. M., Teixeira, M., and Rousseau, D. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9591-9596). The time-resolved step-scan FTIR difference spectra indicate that the rate of decay of the transient Cu(B)1+-CO complex is 34.5 s(-1) and rebinding to heme a(3) occurs with k(2) = 28.6 s(-1). The rate of decay of the transient Cu(B)1+-CO complex displays a similar time constant as the absorption changes at 1694(+)/1706(-), attributed to perturbation of the heme a(3) propionates (COOH). The nu(C-O) of the transient Cu(B)1+-CO species is the same as that of the equilibrium Cu(B)1+-CO species and remains unchanged in the pD range 5.5-9.7 indicating that no structural change takes place at Cu(B) between these states. The implications of these results with respect to proton pathways in heme-copper oxidases are discussed.
authors.affiliation: Department of Chemistry, University of Crete, 71409 Heraklion, Crete, Greece and Paul Scherrer Institut, Life Sciences, OSRA/008, CH-5232 Villigen PSI, Switzerland.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Koutsoupakis
authors.forename: Stavros
authors.initials: S
authors.lastname: Stavrakis
authors.forename: Eftychia
authors.initials: E
authors.lastname: Pinakoulaki
authors.forename: Tewfik
authors.initials: T
authors.lastname: Soulimane
authors.forename: Constantinos
authors.initials: C
authors.lastname: Varotsis
mesh_terms: D000818:Animals; D002244:Carbon / Q000737:chemistry*; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D003573:Cytochrome b Group / Q000737:chemistry*; D003576:Electron Transport Complex IV / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D010100:Oxygen / Q000737:chemistry*; D011522:Protons; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013997:Time Factors
chemical_list: D003573:Cytochrome b Group; D011522:Protons; D002244:Carbon; C058557:cytochrome ba3; D003576:Electron Transport Complex IV; D010100:Oxygen