Year of Award

2017

Document Type

Thesis

Degree Type

Master of Science (MS)

Degree Name

Biochemistry & Biophysics

Department or School/College

Chemistry and Biochemistry

Committee Chair

Dr. Bruce E. Bowler

Committee Co-chair

Dr. J.B. Alexander (Sandy) Ross

Commitee Members

Dr. Bruce E. Bowler, Dr. J. B. Alexander (Sandy) Ross, Dr. D. Scott Samuels, Dr. Scott Miller

Keywords

Cytochrome c, K72A, I81A, Heme Crevice Loop, alkaline conformational transition, proline isomerization

Subject Categories

Biochemistry | Biophysics

Abstract

Cytochrome c, cytc, is a metalloprotein that plays primary roles in electron transport and intrinsic apoptotic pathways. Much of the chemistry that cytc is involved with is regulated by a highly conserved region known as the heme crevice loop, consisting of residues 70-85. Only three of these residues (those at positions 81, 83 and 85) are not universally conserved within the evolutionary timeline. Here I look to elucidate possible evolutionary roles for several of the key residues known to be important in regulating heme chemistry of cytc.

I first address the role that lysine 72 plays in cytc folding and chemistry. Here I provide evidence that K72 alters the alkaline conformational transition of cytc. Trimethylated lysine 72, tmK72, was previously investigated and shows similar trends in peroxidase activity (McClelland et al 2013).

Lastly I address I81 which is not only within the heme crevice loop and not universally conserved, but is also a hydrophobic surface residue. Here I make a Hu I81A variant, mutating to the alanine seen in yeast cytc. Our hypothesis was that this mutation would show a destabilization of the heme crevice loop region when monitoring the charge transfer band (695 nm), and more importantly observing an increase in peroxidase activity when monitoring for tetraguaiacol (470 nm) in our enzymatic assay. This signifies this mutation could have evolved to lock down that heme crevice loop in order to decrease peroxidase activity when intrinsic apoptosis pathways evolved in mammals. pH titration data showed a decrease in stability of the alkaline conformational transition in our I81A variant when compared to Hu WT. When looking at peroxidase activity we see a significant increase in kcat(s-1) values of I81A compared to Hu WT. The Hu I81A indeed shows what we would expect of a mutation which evolved to decrease peroxidase activity. Analysis of pH jump data in the Soret region of cytc shows that there is an effect on lysine 73 or 79 bound alkaline ligands (unable to be determined) by a decrease in amplitude, however there is no effect on the lysine 72 bound alkaline ligand.

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© Copyright 2017 Shiloh M. Nold