A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect

Abstract

A cytochrome c mutant lacking apoptogenic function but competent in electron transfer and antioxidant activities has been constructed. To this end, mutant species of horse and yeast cytochromes c with substitutions in the N-terminal alpha-helix or position 72 were obtained. It was found that yeast cytochrome c was much less effective than the horse protein in activating respiration of rat liver mitoplasts deficient in endogenous cytochrome c as well as in inhibition of H(2)O(2) production by the initial segment of the respiratory chain of intact rat heart mitochondria. The major role in the difference between the horse and yeast proteins was shown to be played by the amino acid residue in position 4 (glutamate in horse, and lysine in yeast; horse protein numbering). A mutant of the yeast cytochrome c containing K4E and some other "horse" modifications in the N-terminal alpha-helix, proved to be (i) much more active in electron transfer and antioxidant activity than the wild-type yeast cytochrome c and (ii), like the yeast cytochrome c, inactive in caspase stimulation, even if added in 400-fold excess compared with the horse protein. Thus this mutant seems to be a good candidate for knock-in studies of the role of cytochrome c-mediated apoptosis, in contrast with the horse K72R, K72G, K72L and K72A mutant cytochromes that at low concentrations were less active in apoptosis than the wild-type, but were quite active when the concentrations were increased by a factor of 2-12.

Computational design of model scaffold for anion recognition based on the ‘CαNN’ motif

“…Characterization of interaction between the respective anion and the designed scaffold for the anion recognition based on the naturally occurring ‘C α NN’ motif segment clearly emphasizes that the interaction is quite localized only at the main chain of the designed anion recognition scaffold segment (for DS1: Gly2‐Lys3‐Ser4; DS2: Gly2‐Gly3‐Ser4; DS3: Ser2‐Lys3‐Ser4 and DS4: Ser2‐Gly3‐Ser4), although there appear a number of Lys residues with suitable side‐chain group ( ) for ionic interaction across the anchoring helix (Figure ). On a closer look one can assign that in each and every interaction irrespective of the anion or the anion recognition scaffold two out of the four oxygen atoms of the anion (sulfate/phosphate) simultaneously interact with the main‐chain C α H, NH, and NH atoms of the respective three consecutive residues of the designed sequence (Figure ), as observed for the crystal structures having the naturally occurring ‘C α NN’ motif [1MUG: residues 107–110; 1YCC: residues 1–4; 1JW9: residues 39–42 and several others] as depicted by Denessiouk et al It is also observed that in each set of the respective 250 docked conformers, out of these two interacting oxygen atoms, one oxygen atom is interacting concurrently with the C α H atom of the i −1th residue and the main chain NH atom of the i th residue; while the other interacting oxygen atom interacts only to the main‐chain NH atom of the i + 1th residue through hydrogen bond (Figure ). This is similar to the reported crystal structures as well as the naturally occurring ‘C α NN’ motif containing context free peptides …”

“…It has been found that among several anion‐recognizing motifs the ‘novel phosphate binding ‘C α NN’ motif’ is different from [Gly/Ser/Thr/Ala‐Gly‐[Gly/Ala] or ‘structural P‐loop’ [(Gly‐Xxx‐Xxx‐Xxx‐Xxx‐Gly‐Lys‐(Ser, Thr)] or ‘nest’ . Unlike other motifs, where the interaction with anion is mediated either via the positively charged side chains and/or via the main chain amides of the anion recognition motif of proteins; participation of the three main chain atoms ( −1 , N i , N i +1 ) of the structurally invariant three consecutive residues at the functional interface and especially the −1 atom in mediating anion‐protein interaction makes the ‘C α NN’ motif unique among the existing other anion binding motifs. It is found that in several occasions this ‘C α NN’ motif having conserved GXX ([Gly/Ser]‐Xaa‐[Thr/Ser]) sequence motif with right handed βαα or βαβ conformation recognizes sulfate ion in lieu of the phosphate ion during crystallization .…”

Conformational change and human cytochrome c function: mutation of residue 41 modulates caspase activation and destabilizes Met-80 coordination

“…The interaction between Apaf-1 and cyt c is thought to be predominantly electrostatic, involving surface lysine residues and the solvent-exposed pyrrole ring C of cyt c [10-15]. It has also been reported that during apoptosis cyt c undergoes a conformational change, which may be required for binding to Apaf-1 [16,17].…”

Conformational Preference of ‘CαNN’ Short Peptide Motif towards Recognition of Anions

“…‘C α NN’ [13], ‘nests’ [14]–[16], ‘structural P-loop’ [17], ‘cup’ [18] along with motifs for recognizing adenine, adenine-containing nucleotides and its analogues [12], [19], [20]], the recently identified ‘C α NN’ motif [13], common to more than 100 fold-representative protein structures as observed in the FSSP database, has a specially characteristic feature. This motif consists of main chain atoms of three consecutive residues (C α −1 N 0 , N +1 ), often present in the active sites of proteins and participates directly in several key regulating functions [1]–[3], [21].…”