Lys-ligated cytochromes make up an emerging family of heme proteins. Density functional theory calculations on the amine/imidazole-ligated c-type ferric heme were employed to develop force-fiel Show more
Lys-ligated cytochromes make up an emerging family of heme proteins. Density functional theory calculations on the amine/imidazole-ligated c-type ferric heme were employed to develop force-field parameters for molecular dynamics (MD) simulations of structural and dynamic features of these proteins. The new force-field parameters were applied to the alkaline form of yeast iso-1 cytochrome c to rationalize discrepancies resulting from distinct experimental conditions in prior structural studies and to provide insights into the mechanisms of the alkaline transition. Our simulations have revealed the dynamic nature of Ω-loop C in the Lys-ligated protein and its unfolding in the Lys-ligated conformer having this loop in the same position as in the native Met-ligated protein. The proximity of Tyr67 or Tyr74 to the Lys ligand of ferric heme iron suggests a possible mechanism of the backward alkaline transition where a proton donor Tyr assists in Lys dissociation. The developed force-field parameters will be useful in structural and dynamic characterization of other native or engineered Lys-ligated heme proteins. Show less
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 c Show more
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 FeIII-X and FeII-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 FeIII-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. Show less
Met80, one of the heme iron ligands in cytochrome c (cyt c), is readily oxidized to Met sulfoxide (Met-SO) by several biologically relevant oxidants. The modification has been suggested to affect both Show more
Met80, one of the heme iron ligands in cytochrome c (cyt c), is readily oxidized to Met sulfoxide (Met-SO) by several biologically relevant oxidants. The modification has been suggested to affect both the electron-transfer (ET) and apoptotic functions of this metalloprotein. The coordination of the heme iron in Met-oxidized cyt c (Met-SO cyt c) is critical for both of these functions but has remained poorly defined. We present electronic absorption, NMR, and EPR spectroscopic investigations as well as kinetic studies and mutational analyses to identify the heme iron ligands in yeast iso-1 Met-SO cyt c. Similar to the alkaline form of native cyt c, Lys73 and Lys79 ligate to the ferric heme iron in the Met80-oxidized protein, but this coordination takes place at much lower pH. The ferrous heme iron is ligated by Met-SO, implying the redox-linked ligand switch in the modified protein. Binding studies with the model peptide microperoxidase-8 provide a rationale for alterations in ligation and for the role of the polypeptide packing in native and Met-SO cyt c. Imidazole binding experiments have revealed that Lys dissociation from the ferric heme in K73A/K79G/M80K (M80K#) and Met-SO is more than 3 orders of magnitude slower than the opening of the heme pocket that limits Met80 replacement in native cyt c. The Lys-to-Met-SO ligand substitution gates ET of ferric Met-SO cyt c with Co(terpy)22+. Owing to the slow Lys dissociation step, ET reaction is slow but possible, which is not the case for nonswitchable M80A and M80K#. Acidic conditions cause Lys replacement by a water ligand in Met-SO cyt c (p Ka = 6.3 ± 0.1), increasing the intrinsic peroxidase activity of the protein. This pH-driven ligand switch may be a mechanism to boost peroxidase function of cyt c specifically in apoptotic cells. Show less