Protein Matrix and Dielectric Effect in Cytochromec

Abstract

The effect of the protein matrix on the standard potential of a buried redox center has been investigated by using a selection of mutants and chemical derivatives in Saccharomyces cerevisiae cytochrome c isoform 1. Assuming only local structural perturbation and no alteration of the iron-ligation chemistry, ⌬E m 0 can be regarded as a measure of the difference in polypeptide solvation of the heme charge, which reflects the dielectric properties of the protein. In double mutants Y67F/N52I Y67F/ N52V, where most of the hydrogen bond network in the heme crevice is eliminated, ⌬S redox compares to the wild type. This indicates that a fully consistent hydrogen bond network has a similar polarizability as an apolar matrix. We therefore argue that the variability in net dielectric susceptibility arises from conformational polarizability, a factor that is not a function of atomic properties and coordinates and is therefore hard to predict using conventional physical relationships.There is still much to learn about the factors determining the E m 0 Ј of redox-active proteins. Determinants of E m 0 Ј such as local structural effects (1) as well as the electrostatic landscape of the polypeptide (2) (including the protein's own charges (3, 4), dipolar matrix (5, 6), and surrounding solvent molecules (7)) have already been identified. However, implementation of this knowledge into models does not reliably reproduce experimental observations.The standard redox potential (E m 0 Ј) reflects the thermodynamics of the equilibrium between redox states. The energetics of rearrangement between redox states will therefore reflect the ability of a system to polarize in an electrostatic field. Assuming that the nature of the iron-ligand interaction remains unchanged, the values of ⌬E m 0 Ј of mutation can be regarded as a measure of change in the polarizability, or dielectric response, to the charge of the redox center.With virtually no large redox-dependent conformational change or ligand rearrangement (8 -12), cytochrome c is a suitable model to investigate the contribution of individual residues to the dielectric properties of the protein. Using three positively charged carboxyamidomethyl-methionine sulfonium ion (CAMMS) 1 derivatives 2 (Fig. 1A) generated from a mutational methionine scan (13), a series of buried mutations at position 52 (Fig. 1B) (14) and the point mutations Y67F, Y67F/ N52I, and Y67F/N52V (15), an attempt to rationalize ⌬E m 0 Ј mut in terms of polarizability of the protein matrix is made. EXPERIMENTAL PROCEDURESDetermination of Redox Potential-The measurement of redox potential was made using the method of mixtures (16) in 50 mM potassium phosphate (pH 7.0). The redox state of the sample protein was assayed in a range of redox buffers set by the ratio of ferro/ferricyanide in solution using an E m 0 Ј for the couple of ϩ0.43 V. Thermodynamics of Oxidoreduction-Temperature dependence of the equilibrium between the reduced and oxidized form of the cytochrome c was measured in 50 mM potassium phosphate (pH 7.0) wit...

Distance dependence of interactions between charged centres in proteins with common structural features

“…Fig. 2 reports typical data for the interaction between surface charged residues for charge reversal and charge neutralisation mutants of Snase [40], barnase and subtilisin [41], for the interaction between charged residues and the haem for iso1‐cytochrome c [42], and in cytochrome c [9], for distances above 6 Å as discussed above. It shows that the magnitude of the electrostatic interactions for surface residues is systematically reduced compared with the expected values from the model presented in this work.…”

“…Comparison of typical published data for interactions between surface charges with the function for distance dependent electrostatic interactions used in this work, and ▵ data from [40], ■ data from [41], • data from [42], and ∘ data from [9].…”

Local stability of Rhodobacter capsulatus cytochrome c2 probed by solution phase hydrogen/deuterium exchange and mass spectrometry

“…For wild-type cytochrome c 2, and Class I c-type cytochromes in general, it is well established that the oxidized state is significantly destabilized relative to the reduced state, a consequence in part of burying a formal charge of plus one (due to the ferric heme iron) in the hydrophobic protein interior [15]. Moreover, it is also clear that the hinge region is significantly destabilized on oxidation with a corresponding increase in its dynamics [9].…”

Cytochrome c: A Multifunctional Protein Combining Conformational Rigidity with Flexibility

“…This perturbation (and thus the splitting) is larger in horse heart than in yeast cytochrome c (Table 1). We measured the cryogenicband spectra of a series of yeast ferrocytochrome c mutants [91] for which Blouin and Wallace had determined the enthalpic and entropic contributions to the redox potential [40]. We could show that the 1 perturbations inferred from the optical spectra correlate nicely with the square of the enthalpic contribution to the redox potential.…”

“…In spite of a rather asymmetric heme environment and strong heme-protein interactions via the two axial ligands [10,30,31], the thioether bridges [32], the internal electric field at the heme [33][34][35][36][37][38], and to a lesser extent substituent-hydrogen bonding [25,26,[39][40][41] and multiple van der Waals contacts [42], spectroscopists have for a long period of time considered the heme as exhibiting an ideal D 4h -symmetry. This was (and in part still is) particularly true for the interpretation of optical absorption and resonance Raman data [43][44][45][46][47][48][49].…”