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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 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. Show less

The hexamer duplex d(CGCGCA).d(TGCGCG) was crystallized with hexammineruthenium(III) ions in an orthorhombic space group; the crystals diffracted to 1.54 A resolution. Strong ion interactions with the adenine base induce a tautomeric shift from the amino to the imino form. Consequently, the A.T base pairing is disrupted. This structural study may be relevant to metal toxicity. Show less

The anticancer activity of cisplatin derives from its ability to bind and cross-link DNA, with the major adduct being the 1,2-d(GpG) intrastrand cross-link. Here, the consequences of this adduct on the conformation, thermal stability, and energetics of duplex DNA are assessed, and the modulation of these parameters by the sequence context of the adduct is evaluated. The properties of a family of 15-mer DNA duplexes containing a single 1,2-d(GpG) cis-¿Pt(NH(3))(2)¿(2+) intrastrand cross-link are probed in different sequence contexts where the flanking base-pairs are systematically varied from T.A to C.G to A.T. By using a combination of spectroscopic and calorimetric techniques, the structural, thermal, and thermodynamic properties of each duplex, both with and without the cross-link, are characterized. Circular dichroism spectroscopic data reveal that the cross-link alters the structure of the host duplex in a manner consistent with a shift from a B-like to an A-like conformation. Thermal denaturation data reveal that the cross-link induces substantial thermal and thermodynamic destabilization of the host duplex. Significantly, the magnitudes of these cross-link-induced effects on duplex structure, thermal stability, and energetics are influenced by the bases that flank the adduct. The presence of flanking A.T base-pairs, relative to T.A or C.G base-pairs, enhances the extent of cross-link-induced alteration to an A-like conformation and dampens the extent of cross-link-induced duplex destabilization. These results are discussed in terms of available structural data, and in terms of the selective recognition of cisplatin-DNA adducts by HMG-domain proteins. Show less