Designed azurins show lower reorganization free energies for intraprotein electron transfer

Abstract

Low reorganization free energies are necessary for fast electron transfer (ET) reactions. Hence, rational design of redox proteins with lower reorganization free energies has been a long-standing challenge, promising to yield a deeper understanding of the underlying principles of ET reactivity and to enable potential applications in different energy conversion systems. Herein we report studies of the intramolecular ET from pulse radiolytically produced disulfide radicals to Cu(II) in rationally designed azurin mutants. In these mutants, the copper coordination sphere has been fine-tuned to span a wide range of reduction potentials while leaving the metal binding site effectively undisrupted. We find that the reorganization free energies of ET within the mutants are indeed lower than that of WT azurin, increasing the intramolecular ET rate constants almost 10-fold: changes that are correlated with increased flexibility of their copper sites. Moreover, the lower reorganization free energy results in the ET rate constants reaching a maximum value at higher driving forces, as predicted by the Marcus theory.cupredoxin | Type 1 copper | blue copper | reorganization energy | Marcus inverted region E lectron transfer (ET) through proteins is central to biological energy conversion processes, from photosynthesis to respiration (1). ET rates between redox centers are tightly controlled and tuned in biological systems for efficiency and to avoid formation of deleterious products (2-5). An important parameter determining the ET rate is the reorganization free energy of the redox sites (6-8), which for biochemical ET reactions has evolved to low values, usually providing high rate constants. Despite many years of work, few efforts have succeeded in designing protein ET sites having reorganization free energies lower than those observed for the native ones. For example, numerous mutations proximal to the type 1 (T1) blue copper site have been introduced into azurin (Az) (9-19), a bacterial electron-mediating protein, but those that have lowered this barrier have not been reported yet.Prominent among the theories developed to rationalize the control of ET reaction rates (20-22) is that of R. A. Marcus (23). A particularly intriguing element of this theory predicts that the ET rate reaches its maximum when the driving force of the reaction equals the reorganization free energy, after which an even higher driving force decreases ET rate, reaching the "Marcus inverted region." It has been proposed that the dramatic difference in rates between some of the forward and backward ET reactions in the photosynthetic reaction center is a result of the latter being in the inverted region, which is reached because of exceptionally low reorganization free energies of the relevant ET sites (24).In the present study, we aim at examining how the major changes in modifying the redox potential of the type 1 copper site in Az achieved by Marshall et al. (25) affect its ET reactivity. A major obstacle in rationally fine-tuning the properties of t...

Protein Design: Toward Functional Metalloenzymes

“…Furthermore, this work showed varied intraprotein electron transfer capability of certain mutants, which is indicative of variation of the reorganization energy with rational design. 393 …”

“…Farver et al 393 measured the electron transfer rates to address the question of whether or not a large range of redox potentials can also lead to controlled variation of the intramolecular electron transfer rates. Pulse radiolytically induced CO 2 − radical anions were introduced to these anaerobic Az derivatives to reduce the disulfide bond, located at the opposite side of Az relative to the copper center (Figure 11A), into a disulfide radical anion.…”

“…Mutants: 1, Phe114Pro/Met121Gln; 2, Phe114Pro; 3, Asn47Ser/Phe114Asn; 4, Asn47Ser/Met121Leu; 5, Phe114Asn/ Met121Leu; 6, Asn47Ser/Phe114Asn/Met121Leu. Reproduced with permission from ref 393. Copyright 2013 National Academy of Sciences.…”

Sulfonamido tripods: Tuning redox potentials via ligand modifications

“…In series of studies, Lu showed that changes in the primary and secondary coordination spheres of a single Cu center had a large impact on the Cu I/ Cu II redox process in blue copper proteins. 4–6 Relatively small changes in redox potentials resulted when a tyrosine residue near the Fe III center in rubredoxin was modified with nonnative tyrosine residues with differing para -substituents. 7 Analysis of electrochemical measurements did however demonstrate strong correlation between the reduction potential of the Fe III center and the Hammett constants (σ p ) of the para -substituent of the nonnative tyrosine-based residues.…”

Long-Range Electron Transfer in Engineered Azurins Exhibits Marcus Inverted Region Behavior

“…37 In a previous study, we have attributed such lowering of the reorganization energy to increased flexibility of the T1 copper center caused by changes in noncovalent interactions such as hydrogen bonding and hydrophobicity in the secondary coordination sphere of the T1 copper site. 43 It has been shown before that changing hydrogen bonds and collective perturbation of the protein dynamics can affect the λ values. 48 The deviations from the fit line at the highest driving force would, if attributed only to variation in the reorganization energy, correspond to a variation of up to 0.25 eV from the average or fit value.…”