Kun-Yun Yang

PhD : 2006
Fellow Researcher
Caltech

 

My Story:

Thesis title:
The effects of the N(5) hydrogen bond and the re-face positive charge on the redox properties of flavin in the methylotrophic bacterium W3A1 electron transfer flavoprotein

Abstract:
In flavoproteins, the interactions between the flavin cofactor and its protein environment modulate the physical and chemical properties of the flavin, which is critical for the biological functions of flavoproteins. In this thesis, the electron transfer flavoprotein from methylotrophic bacterium W3A1 (wETF) is used to study the flavin-protein interactions and their roles in the modulation of the redox properties of its flavin cofactor. First, Raman spectroscopy has been used to investigate flavin-protein interactions in the oxidized wETF. Several unique features of wETF were investigated based on the Raman data of wETF in the oxidized state. For the first time, high quality non-resonance Raman spectra for the SQ- form of the flavin, as stabilized by the wETF protein, have been obtained. In addition, the first normal mode analysis of flavin SQ- form was carried out in this study. The effects of the change of the N(5) H-bond on the Raman spectrum of the flavin SQ- form was evaluated. Second, the effects of the H-bond at the N(5) of the flavin isoalloxazine ring at the oxidized state on the Eox/sq of the flavin have been studied in the wETF system. The strength of N(5) H-bond in wETF was systematically altered by the substitution of aS254 residue with Thr, Cys and Ala. In all three mutants, the Eox/sq is significantly affected and show correlation with the strength of N(5) H-bond as indicated by Raman spectra, suggesting that the N(5) H-bond plays a role in the modulation of the Eox/sq of flavin. Third, the role of the positive charge in the re-face of the flavin in the modulation of the Eox/sq has been investigated by a serial of mutations on the aR237 residue in this position for systematically disturbing, neutralizing, reversing the positive charge of this position. The Eox/sq were dramatically affected by the substitutions of aR237, and show a correlation with the charge and the position of the residue in the position. These results demonstrated that the positive charge and its position are responsible for the highly stable anionic semiquinone state and the unusually high Eox/sq of wETF.

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