Distal Unfolding of Ferricytochrome c Induced by the F82K Mutation

It is well known that axial coordination of heme iron in mitochondrial cytochrome c has redox-dependent stability. The Met80 heme iron axial ligand in the ferric form of the protein is relatively labile and can be easily replaced by alternative amino acid side chains under non-native conditions induced by alkaline pH, high temperature, or denaturing agents. Here, we showed a redox-dependent destabilization induced in human cytochrome c by substituting Phe82—conserved amino acid and a key actor in cytochrome c intermolecular interactions—with a Lys residue. Introducing a positive charge at position 82 did not significantly affect the structure of ferrous cytochrome c but caused localized unfolding of the distal site in the ferric state. As revealed by 1H NMR fingerprint, the ferric form of the F82K variant had axial coordination resembling the renowned alkaline species, where the detachment of the native Met80 ligand favored the formation of multiple conformations involving distal Lys residues binding to iron, but with more limited overall structural destabilization.

A molecular method for a qualitative analysis of potentially coding sequences of DNA

Total sequence phylogenies have low information content. Ordinary misconceptions are that character quality can be ignored and that relying on computer algorithms is enough. Despite widespread preference for a posteriori methods of character evaluation, a priori methods are necessary to produce transformation series that are independent of tree topologies. We propose a stepwise qualitative method for analyzing protein sequences. Informative codons are selected, alternative amino acid transformation series are analyzed, and most parsimonious transformations are hypothesized. We conduct four phylogenetic analyses of philodryanine snakes. The tree based on all nucleotides produces least resolution. Trees based on the exclusion of third positions, on an asymmetric step matrix, and on our protocol, produce similar results. Our method eliminates noise by hypothesizing explicit transformation series for each informative protein-coding amino acid. This approaches qualitative methods for morphological data, in which only characters successfully interpreted in a phylogenetic context are used in cladistic analyses. The method allows utilizing character information contained in the original sequence alignment and, therefore, has higher resolution in inferring a phylogenetic tree than some traditional methods (such as distance methods).