Skip to main content

Flavin semiquinone stability and evolution of function in photolyases and cryptochromes

Resource type
Thesis type
(Thesis) Ph.D.
Date created
This thesis examines the mechanism under which Photolyases (PLs) and Cryptochromes (CRYs) tune their semiquinone stability and reactivity and correlate these properties with function. The work is supported by experiments in site-directed mutagenesis, oxidation kinetics, isotope effects, electrochemistry and in vivo cellular functional assays. I present the first evaluation of both flavin redox potentials for PL, which establish, counter to existing models, that its semiquinone radical is thermodynamically destabilized. Investigations of the N5-proximal Asn-to-Asp mutant decreases the driving force for oxidation but alleviates the kinetic barrier. Analysis of additional mutants demonstrates that the modulation at the N5-proximal residue is context dependent. These results support a regulatory mechanism that exploits proton-coupled electron transfer and changes in protein conformation to adjust the kinetic barriers to redox reactions of the semiquinone in PLs and CRYs. I also provide evidence for the biological relevance of redox tuning by correlating semiquinone stability to in vivo DNA repair by a series of PL mutants exhibiting a varying range of stability. Together, these investigations provide molecular-level rationale for the evolution of function in PLs and CRYs. My results also underscore two emerging general themes in biological electron transfer, the significance of proton transfer and kinetic mechanisms of redox regulation.
Copyright statement
Copyright is held by the author.
The author granted permission for the file to be printed and for the text to be copied and pasted.
Scholarly level
Supervisor or Senior Supervisor
Thesis advisor: Pinto, B. Mario
Member of collection
Download file Size
etd7042_MDamiani.pdf 49.35 MB

Views & downloads - as of June 2023

Views: 0
Downloads: 0