In the previous chapter, we have generated atomic coordinates of a synthetic protein recently synthesized (SynCytó) to model the central part of the native cytochrome b (Cytó) subunit consisting of a four-helix bundle with two hemes as cofactors. Since the X-ray structure is not solved, the structural elements of SynCytó were assembled from scratch using all known chemical and structural information available, thereby avoiding strain and atomic clashes. The molecular mechanics was subsequently employed to fully relax the model structure of SynCytó by applying the stepwise constraint energy minimization. Molecular dynamics simulations are applied to the computer-generated synthetic protein structure and the crystal structure of native Cytó, revealing very similar root-mean-square deviation (RMSD) profiles over time and, thus, demonstrating a relative rigidness, stability, and strain-free of the finally modeled structure. In this chapter, to further test and validate the computer-generated structure of SynCytó, characteristic observables have to be computed and compared with measured quantities. One of these is the redox potential, a key parameter of redox-active proteins. The results of the redox potentials calculation are discussed here. For comparison, protonation patterns and heme redox potentials in the SynCytó model structure and native Cytó from the respiratory cytochrome bc¡ (Cytbci) complex are calculated from the electrostatic energies by solving the Poisson-Boltzmann equation. The computed redox potentials generally agree within 20 mV with the experimental measurements. The factors of protein environment and different energy terms determining a shift in the heme redox potentials are elucidated and analyzed in detail. In addition, the pH dependence of the redox potential of all hemes is evaluated in the physiologically relevant pH range. This chapter also rationalizes the effects of the applied ionic strength, external redox potential of bulk solvent (aqueous solution), the coupling of protonation and reduction reactions, and the distribution and population of the microscopic redox states in the studied biomolecular systems. The redox-Bohr effect in native Cytó is modeled successfully, whereby the essential protonatable groups responsible fortius phenomenon are identified. A good agreement between calculated and measured redox potentials offers the possibility to analyze the factors of the protein environment determining the shift in the redox potentials of hemes, which are cofactors in the synthetic and native cytochromes. That opens new avenues to understand how nature tunes the redox potentials of cofactors in proteins to perform their biological functions and how one can construct synthetic proteins whose cofactors have desired redox potentials. The applied approach is valuable in studying structure-function correlation and understanding protein-cofactor interactions in atomic detail.
Dragan Popović (Sat,) studied this question.