Conformational dynamics coupled to protonation equilibrium at the CuA site of Thermus thermophilus: insights into the origin of thermostability

Biochemistry
Nusrat J M Sanghamitra, Shyamalava Mazumdar

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...Continue Reading

References

Dec 18, 1991·European Journal of Biochemistry·R Jaenicke
Nov 1, 1990·Quarterly Reviews of Biophysics·M Saraste
Jan 1, 1974·Journal of Biochemistry·T Oshima, K Imahori
Jan 1, 1996·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·R Jaenicke
Feb 3, 2005·Proteins·Han-Kuen LiangJenn-Kang Hwang

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Citations

Dec 24, 2009·The Journal of Biological Chemistry·Stephanie L GanttCarol A Fierke
May 9, 2013·Chemical Communications : Chem Comm·Damián Alvarez-PaggiAlejandro J Vila
Dec 14, 2018·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Taylor DevlinLaura M Hunsicker-Wang

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