Probing Protonation/Deprotonation of Tyrosine Residues in Cytochrome ba3 Oxidase from Thermus thermophilus by Time-resolved Step-scan Fourier Transform Infrared Spectroscopy

Constantinos Koutsoupakis,Olga Kolaj-Robin,Tewfik Soulimane,Constantinos Varotsis

doi:10.1074/jbc.m111.252213

Constantinos Koutsoupakis, Olga Kolaj-Robin + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m111.252213

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Abstract

Elucidating the properties of the heme Fe-Cu(B) binuclear center and the dynamics of the protein response in cytochrome c oxidase is crucial to understanding not only the dioxygen activation and bond cleavage by the enzyme but also the events related to the release of the produced water molecules. The time-resolved step-scan FTIR difference spectra show the ν(7a)(CO) of the protonated form of Tyr residues at 1247 cm(-1) and that of the deprotonated form at 1301 cm(-1). By monitoring the intensity changes of the 1247 and 1301 cm(-1) modes as a function of pH, we measured a pK(a) of 7.8 for the observed tyrosine. The FTIR spectral changes associated with the tyrosine do not belong to Tyr-237 but are attributed to the highly conserved in heme-copper oxidases Tyr-136 and/or Tyr-133 residue (Koutsoupakis, K., Stavrakis, S., Pinakoulaki, E., Soulimane, T., and Varotsis, C. (2002) J. Biol. Chem. 277, 32860-32866). The oxygenation of CO by the mixed-valence form of the enzyme revealed the formation of the ∼607 nm P (Fe(IV)=O) species in the pH 6-9 range and the return to the oxidized form without the formation of the 580 nm F form. The data indicate that Tyr-237 is not involved in the proton transfer pathway in the oxygenation of CO by the mixed-valence form of the enzyme. The implication of these results with respect to the role of Tyr-136 and Tyr-133 in proton transfer/gating along with heme a(3) ring D propionate-H(2)O-ring A propionate-Asp-372 site to the exit/output proton channel (H(2)O pool) is discussed.

Highlights

Cans and bovine oxidase despite the fact that most of the residues belonging to these pathways are not conserved [1]
It has been suggested that the additional electron needed to produce the 607 nm P (Fe(IV)ϭO) species is provided by Tyr-167 or Trp-272 (P. denitrificans sequence), which are near the binuclear heme Fe-CuB center and highly conserved in heme-copper oxidases (Tyr-136 and Trp-229 in T. thermophilus) [16, 17]
PKa of the Tyrosine Residue—Intensity changes and frequency shifts of side chains and backbone structures have been observed in the FTIR difference spectra of heme-copper oxidases as the result of an electrochemical perturbation of the metal centers at room temperature or of the perturbation induced by the photodissociation of CO bound to the heme at 80 K, at which the CO binds irreversibly to CuB [18, 19, 23, 24]

Summary

Introduction

Cans and bovine oxidase despite the fact that most of the residues belonging to these pathways are not conserved [1]. In the work presented here, we continued our TRS2-FTIR approach with heme-copper oxidases at room temperature, and in conjunction with the assignment of ␯7a(CO) and ␦(COH) in the aa3-type oxidase from P. denitrificans, we detected the protonated and deprotonated forms of tyrosine residue(s) near the induced perturbation, i.e. the heme a3-CuB binuclear center of cytochrome ba3 from T. thermophilus [4, 5, 17, 18, 23, 24].

Results

Conclusion