Comparing Properties of Common Bioinorganic Ligands with Switchable Variants of Cytochromec

Abstract

Ligand substitution at the metal center is common in catalysis and signal transduction of metalloproteins. Understanding the effects of particular ligands, as well as the polypeptide surrounding, is critical for uncovering mechanisms of these biological processes and exploiting them in the design of bioinspired catalysts and molecular devices. A series of switchable K79G/M80X/F82C (X = Met, His, or Lys) variants of cytochrome (cyt) c was employed to directly compare the stability of differently ligated proteins and activation barriers for Met, His, and Lys replacement at the ferric heme iron. Studies of these variants and their nonswitchable counterparts K79G/M80X have revealed stability trends Met < Lys < His and Lys < His < Met for the protein Fe III −X and Fe II −X species, respectively. The differences in the hydrogen-bonding interactions in folded proteins and in solvation of unbound X in the unfolded proteins explain these trends. Calculations of free energy of ligand dissociation in small heme model complexes reveal that the ease of the Fe III −X bond breaking increases in the series amine < imidazole < thioether, mirroring trends in hardness of these ligands. Experimental rate constants for X dissociation in differently ligated cyt c variants are consistent with this sequence, but the differences between Met and His dissociation rates are attenuated because the former process is limited by the heme crevice opening. Analyses of activation parameters and comparisons to those for the Lys-to-Met ligand switch in the alkaline transition suggest that ligand dissociation is entropically driven in all the variants and accompanied by Lys protonation at neutral pH. The described thiolate redox-linked switches have offered a wealth of new information about interactions of different protein-derived ligands with the heme iron in cyt c model proteins, and we anticipate that the strategy of employing these switches could benefit studies of other redox metalloproteins and model complexes.

Naturally Occurring I81N Mutation in Human Cytochrome c Regulates Both Inherent Peroxidase Activity and Interactions with Neuroglobin

“…Previous studies have shown that mutations in the Ω-loop D (70–85) such as K72A, P76C, K79G/M80X, I81A, F82K, and V83G may alter the heme crevice dynamics and ligand-binding properties. 19 , 32 , 40 − 42 …”

“…The faster rate of the azide binding to the heme of I81N h Cyt c may suggest the faster dissociation of the Met80-heme ligation. Previous studies have shown that mutations in the Ω-loop D (70–85) such as K72A, P76C, K79G/M80X, I81A, F82K, and V83G may alter the heme crevice dynamics and ligand-binding properties. ,,− …”

Influence of the Interdomain Interface on Structural and Redox Properties of Multiheme Proteins

“…The unfolding profiles for the M66H and M167H in both ferric and ferrous states display two separate transitions (Figure ). Studies of M80H cyt c have revealed that the stability of the His-ligated protein decreases only slightly in the ferric state, but drops dramatically in the ferrous state compared to the stability of the Met-ligated WT . With this knowledge, we assign the first unfolding transition to be of the c 4 -B domain in both ferric variants.…”

“…Studies of M80H cyt c have revealed that the stability of the His-ligated protein decreases only slightly in the ferric state, but drops dramatically in the ferrous state compared to the stability of the Met-ligated WT. 36 With this knowledge, we assign the first unfolding transition to be of the c 4 -B domain in both ferric variants. Assuming then that the His-ligation does not change much the stability of ferric c 4 -A and c 4 -B, the unfolding order of the two domains in M66H and M167H is the same as that in ferric WT c 4 .…”

“…Two variants of the full-length WT c 4 , M66H and M167H, with the Met residue corresponding to the axial ligand at Heme A or Heme B replaced by a His residue, were prepared. Studies of cyt c have established that the replacement of the Met ligand to His leads to a decrease in the reduction potential of the heme iron and alters the stability of the protein. ,, …”