Redox-dependent stability,protonation, and reactivity of cysteine-bound heme proteins

Cysteine-bound hemes are key components of many enzymes and biological sensors. Protonation (deprotonation) of the Cys ligand often accompanies redox transformations of these centers. To characterize these phenomena, we have engineered a series of Thr78Cys/Lys79Gly/Met80X mutants of yeast cytochrome c (cyt c) in which Cys78 becomes one of the axial ligands to the heme. At neutral pH, the protonation state of the coordinated Cys differs for the ferric and ferrous heme species, with Cys binding as a thiolate and a thiol, respectively. Analysis of redox-dependent stability and alkaline transitions of these model proteins, as well as comparisons to Cys binding studies with the minimalist heme peptide microperoxidase-8, demonstrate that the protein scaffold and solvent interactions play important roles in stabilizing a particular Cys–heme coordination. The increased stability of ferric thiolate compared with ferrous thiol arises mainly from entropic factors. This robust cyt c model system provides access to all four forms of Cys-bound heme, including the ferric thiol. Protein motions control the rates of heme redox reactions, and these effects are amplified at low pH, where the proteins are less stable. Thermodynamic signatures and redox reactivity of the model Cys-bound hemes highlight the critical role of the protein scaffold and its dynamics in modulating redox-linked transitions between thiols and thiolates.Iron–cysteine bonds are common in biological systems, especially in heme enzymes and sensors. P450 enzymes, with a negatively charged Cys thiolate coordinated to the heme, catalyze diverse oxidation reactions and are important targets both for therapeutic intervention and industrial catalysis (1–3). Heme–thiolate nitric oxide synthase (NOS) (4, 5) and cystathionine β-synthase (CBS) (6) are responsible for the formation of the signaling molecule nitric oxide and detoxification of homocysteine, respectively. Two other heme-thiolate enzymes, chloroperoxidase (7) and the SoxAX complex (8) play important roles in the synthesis of halogenated compounds and oxidation of thiosulfate and sulfide, respectively. Thiolate-bound hemes are also found in many sensor proteins (9), including those that regulate circadian rhythms in mammals (10).The protonation state of the coordinated Cys is critical for the catalytic function of these enzymes and ligand lability of the sensors. The deprotonated Cys provides a strong thiolate “push” that enables heterolytic O—O bond cleavage by P450 enzymes (1). Protonation of the native thiolate ligand to a neutral thiol has been suggested as a mechanism of P450 deactivation yielding the infamous P420 species (11). The ferrous thiol is easily displaced by other ligands resulting in functional conformational changes in sensors (9, 12) but also, upon exposure to dioxygen (13), in deleterious effects of Cys oxidation.Protonation (or deprotonation) reactions of a Cys ligand often accompany redox transformations of the Cys-bound hemes. Heme reduction increases the electron density on the iron increasing the effective pK_a of the coordinated thiol. Although ferrous thiol coordination is frequently lost, model studies have indicated that neutral Cys is a viable ligand to the ferrous heme (12). For ferric hemes, where the metalloporphyrin dianion unit has a core charge of +1, a thiolate is the preferred ligand. Only a few examples of thiol-ligated ferric hemes are currently known (14–16), all with highly electron-rich systems.Understanding redox-dependent stability and protonation of Cys-bound hemes is critical for establishing mechanistic principles of these redox centers. With native systems, their evolved function often limits the number of easily observed species. Small synthetic models (14, 17) are useful for detailed thermodynamic and kinetic investigations but may not capture all of the complexity of the protein framework. Finally, reduction of the heme iron and degradation of the porphyrin by thiyl radicals complicate studies at high concentration of thiols (18, 19).In the present study, we have engineered a series of Thr78Cys/Lys79Gly/Met80X (X = Leu, Ile, or Phe) mutants of yeast cytochrome c (cyt c) in which the original Met80 ligand was mutated to noncoordinating residues and Cys78 becomes one of the axial ligands to the heme (Fig. 1). The strategic placement of a coordinating Cys in the hydrophobic interior of this protein has yielded a robust system for examining redox-dependent stability and interconversions of Cys-bound hemes. This model system allows a detailed thermodynamic characterization of ferric thiolate and ferrous thiol species and also provides access to kinetic intermediates. The results demonstrate the importance of the polypeptide scaffold for redox-dependent stability of Cys-bound heme proteins and highlight the role of protein motions in their redox reactions.Open in a separate windowFig. 1.(A) Structure of yeast iso-1 cyt c (2YCC) (54) showing positions of mutated residues. (B) EPR spectra at 10 K of ferric Thr78Cys/Lys79Gly/Met80X variants of yeast iso-1 cyt c and Met80Cys variant of horse heart cyt c in a 100 mM sodium phosphate buffer at pH 7.4.