Fold versus sequence effects on the driving force for protein‐mediated electron transfer

Abstract

Electron transport chains composed of electron transfer reactions mainly between proteins provide fast, efficient flow of energy in a variety of metabolic pathways. Reduction potentials are essential characteristics of the proteins because they determine the driving forces for the electron transfers. Since both polar and charged groups from the backbone and side chains define the electrostatic environment, both the fold and the sequence will contribute. However, while the role of a specific sequence may be determined by experimental mutagenesis studies of reduction potentials, understanding the role of the fold by experiment is much more difficult. Here, continuum electrostatics and density functional theory calculations are used to analyze reduction potentials in [4Fe-4S] proteins. A key feature is that multiple homologous proteins in three different folds are compared: six high potential iron-sulfur proteins, four bacterial ferredoxins, and four nitrogenase iron proteins. Calculated absolute reduction potentials are shown to be in quantitative agreement with electrochemical reduction potentials. Calculations further demonstrate that the contribution of the backbone is larger than that of the side chains and is consistent for homologous proteins but differs for non-homologous proteins, indicating that the fold is the major protein factor determining the reduction potential while the specific amino acid sequence tunes the reduction potential for a given fold. Moreover, the fold contribution is determined mainly by the proximity of the redox site to the protein surface and the orientation of the dipoles of backbone near the redox site.

Density functional theory calculations of redox properties of iron–sulphur protein analogues

“…This 'DFT þ PB' approach may be considered a zeroth-order solution that ignores the interactions between the inner and outer spheres other than the classical interactions between the partial charges of the outer sphere and the inner sphere in the two oxidation states. Although this approach may seem crude, our DFT þ PB calculations of reduction potentials are in excellent agreement with electrochemical measurements [29]. Moreover, deviations from the zeroth-order solution allow us to identify unique interactions not accounted for in the simple picture.…”

“…On the other hand, our calculations also showed that when the thiolate ligands have dihedral angles corresponding to Fd, localisation of the minority spin is induced on one layer (Figure 13), leading to a large decrease in %L with only small changes in the redox energy when the thiolate ligands have dihedral angles corresponding to HiPIP, which has the typical delocalised minority spin for both redox layers. These findings suggest that the conformations of the cysteinyl ligands give rise to large changes in %L between the Fds and the HiPIPs but only contribute ,100 mV to the differences in reduction potential between the two, consistent with the large contributions from the surrounding protein calculated by DFT þ PB [29].…”

“…calculations of gas-phase redox site analogues in which the cysteine ligands are replaced by methylthiolates ( Figure 1) and the outer sphere energies by PoissonBoltzmann (PB) continuum electrostatics [26,27] calculations using X-ray crystallographic structures of proteins [28,29]. This 'DFT þ PB' approach may be considered a zeroth-order solution that ignores the interactions between the inner and outer spheres other than the classical interactions between the partial charges of the outer sphere and the inner sphere in the two oxidation states.…”

“…The calculations for several homologues of three types of [4Fe -4S] proteins, the high potential iron-sulfur proteins (HiPIPs), the ferredoxins (Fds) and the iron-protein of nitrogenase, show excellent correlation with experimental reduction potentials (Figure 9). This has allowed studies that demonstrate how the redox site, the fold and the sequence contribute to the net reduction potential of a redox protein [29]. In addition, applications for finding mutations that alter reduction potentials, locating residues that cause pH-dependent reduction potentials, and even evaluating dimer interactions of redox active potentials are being developed.…”

Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity

“…The clusters of both BSSβ and BSSγ do appear to play a structural role in that their removal leads to dissociation of the subunits from BSSα (16), which in turn leaves BSSα prone to unfolding and clearance by cellular degradation machinery in vivo (16) and sensitive to proteases in vitro. In terms of whether these clusters also play a redox role in addition to the above structural role, we find that the overall fold of both subunits is derived from a HiPIP, a family of [4Fe-4S] cluster proteins known for their high redox potentials (100-450 mV) and for their ability to stabilize clusters in the +3 oxidation state (20,27). A universal pattern of hydrophobic shielding of the [4Fe-4S] clusters in HiPIPs is believed to be responsible for shifting the +3/+2 redox potential into a biologically relevant range and shifting the +2/+1 couple out of a biological window, with potentials of -1.75 V (28).…”