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

Konstantinos Koutsoupakis, Stavros Stavrakis, Eftychia Pinakoulaki, Tewfik Soulimane, Constantinos Varotsis

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Abstract

We report the first evidence for the existence of the equilibrium CuB1+-CO species of CO-bound reduced cytochrome ba3 from Thermus thermophilus at room temperature. The frequency of the C-O stretching mode of CuB1+-CO is located at 2053 cm-1 and remains unchanged in H2O/D2O exchanges and, between pD 5.5 and 9.7, indicating that the chemical environment does not alter the protonation state of the CuB histidine ligands. The data and conclusions reported here are in contrast to the changes in protonation state of CuB-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 CUB1+-CO complex is 34.5 s-1 and rebinding to heme a3 occurs with k2 = 28.6 s-1. The rate of decay of the transient CuB1+-CO complex displays a similar time constant as the absorption changes at 1694(+)/1706(-), attributed to perturbation of the heme a3 propionates (COOH). The ν(C-O) of the transient CuB1+-CO species is the same as that of the equilibrium CuB1+-CO species and remains unchanged in the pD range 5.5-9.7 indicating that no structural change takes place at CuB between these states. The implications of these results with respect to proton pathways in heme-copper oxidases are discussed.

Original languageEnglish
Pages (from-to)32860-32866
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number36
DOIs
Publication statusPublished - 6 Sep 2002
Externally publishedYes

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