title: Conformational dynamics coupled to protonation equilibrium at the CuA site of Thermus thermophilus: insights into the origin of thermostability.
abstract: An extremely slow pH dependent conformational equilibrium between a valence-delocalized and a valence-trapped species of the dinuclear CuA domain of cytochrome c oxidase from Thermus thermophilus has been identified and characterized using UV-visible absorption, circular dichroism, time-resolved fluorescence and electron paramagnetic resonance spectroscopy as well as by stopped-flow kinetic techniques. The results indicated that the nature of this pH dependent conformation change in the CuA domain in the Thermus protein was distinctly different from that observed in the mesophilic analogue from Paracoccus denitrificans and in the engineered CuA domain in azurin. pH jump kinetic studies suggested existence of a fast deprotonation equilibrium followed by slow conformational change in the protein, which is contrary to that observed in the case of the analogous protein from P. denitrificans. Continuous-flow electrospray mass spectral studies on H/D exchange in the TtCuA showed that approximately 75% of the protons are exchanged within the dead-time of the experiment supporting fast proton transfer kinetics in the protein. Analysis of temperature dependence of the kinetics of the conformational transition showed that the rigidity of the protein structure decreases with increase in temperature. The results indicated that though the rate of proton transfer at individual sites in the protein could be very fast, the conformational change that requires simultaneous breaking of several interactions in a segment of the structure might be slow in the thermostable protein.
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.
authors.forename: Nusrat J M
authors.initials: NJ
authors.lastname: Sanghamitra
authors.forename: Shyamalava
authors.initials: S
authors.lastname: Mazumdar
mesh_terms: D003300:Copper / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry*; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology
chemical_list: D003300:Copper; D003576:Electron Transport Complex IV