2 research outputs found
EVOLUTION OF THE KINETICS AND DYNAMICS OF HEME-CREVICE LOOP REGULATING CHEMISTRY IN HUMAN CYTOCHROME C
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
Effect of a K72A Mutation on the Structure, Stability, Dynamics, and Peroxidase Activity of Human Cytochrome <i>c</i>
We
test the hypothesis that Lys72 suppresses the intrinsic peroxidase
activity of human cytochrome <i>c</i>, as observed previously
for yeast iso-1-cytochrome <i>c</i> [McClelland, L. J.,
et al. (2014) <i>Proc. Natl. Acad. Sci. U. S. A. 111</i>, 6648–6653]. A 1.25 Å X-ray structure of K72A human
cytochrome <i>c</i> shows that the mutation minimally affects
structure. Guanidine hydrochloride denaturation demonstrates that
the K72A mutation increases global stability by 0.5 kcal/mol. The
K72A mutation also increases the apparent p<i>K</i><sub>a</sub> of the alkaline transition, a measure of the stability of
the heme crevice, by 0.5 unit. Consistent with the increase in the
apparent p<i>K</i><sub>a</sub>, the rate of formation of
the dominant alkaline conformer decreases, and this conformer is no
longer stabilized by proline isomerization. Peroxidase activity measurements
show that the K72A mutation increases <i>k</i><sub>cat</sub> by 1.6–4-fold at pH 7–10, an effect larger than that
seen for the yeast protein. X-ray structures of wild type and K72A
human cytochrome <i>c</i> indicate that direct interactions
of Lys72 with the far side of Ω-loop D, which are seen in X-ray
structures of horse and yeast cytochrome <i>c</i> and could
suppress peroxidase activity, are lacking. Instead, we propose that
the stronger effect of the K72A mutation on the peroxidase activity
of human versus yeast cytochrome <i>c</i> results from relief
of steric interactions between the side chains at positions 72 and
81 (Ile in human vs Ala in yeast), which suppress the dynamics of
Ω-loop D necessary for the intrinsic peroxidase activity of
cytochrome <i>c</i>