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

Abstract

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.metalloenzyme | folding | electron transfer

Engineered holocytochrome c synthases that biosynthesize new cytochromes c

“…3) (Table S3), and are reflective of the presence of axial His/Met ligands. The EPR of yeast iso-1 cyt c has been recorded for Met81 variants: Asp, Ala, Cys, Leu, Lys, Ile, and Phe (21,22). Of these, yeast iso-1 cyt c M81A shows the most similarity to the human cyt c M81A signals, which exhibit g values at 2.58, 2.18, and 1.86 (Fig.…”

Reactivity of Inorganic Sulfide Species toward a Heme Protein Model

“…A recent publication about the reactivity of cysteine toward engineered cytochrome c mutants and NAcMP8 highlights the versatile role of the protein environment in the speciation of the ligand and the redox stability of the heme. 65 In summary, Fe III NAcMP11 provides a model that is devoid of distal mechanisms of stabilization and kinetic barriers imposed by a protein structure, for the evaluation of proximal effects on the formation of a hexacoordinated complex with a sulfide species as the sixth ligand. The formation of a moderately stable, ferric low-spin species coordinated to a form of sulfide, characterized by a Soret maximum at 414 nm (P 414 ), is tentatively assigned to NAcMP11Fe III (SH 2 /SH − ) from the spectroscopic and kinetic data.…”

Lead discovery and in silico 3D structure modeling of tumorigenic FAM72A (p17)

“…In the context of CASP's assessment [56][57][58] for the advancement of methods of identifying 3D protein structures from their AA sequences, we observed that the template-based (3GA3_A) 3D FAM72A protein structure (with an overall Gfactor=−0.2) has a much reliable overall stereochemical and Binding interactions of FAM72A with Zn 2+ , Fe 3+ , and nucleic acid indicate that this protein may have a significant cellular function as a transcription factor, a finding that might be particularly interesting in view of FAM72A's role in cancer cell signaling [3] and may also interfere with enzymatic redox reactions [59][60][61].…”