A single protein redox ruler.

Redox reactions are the heart of function in biological systems. Using a deceptively simple toolbox, consisting largely of first row transition metals and the 20 canonical amino acids, nature has evolved proteins that contain cofactors with reduction potentials (E°′) between +1 V and −1 V. Chemists have long known that ligands to transition metals markedly alter E°′ of the metal, often in a systematic way. A set of such trends is much less clear in metalloproteins. In PNAS, Hosseinzadeh et al. (1) used azurin, a blue copper protein from Pseudomonas aeruginosa, as a framework that can be rationally modified to shift E°′ of the embedded metal site between +0.97 V and −0.95 V [all potentials are with respect to the normal hydrogen electrode (NHE)]. Tuning E°′ over such a large range was made possible using five (or fewer) nature-inspired mutations and substitution with Cu or Ni. Copper sites carry out many redox roles in biological systems. Functions of type 1 sites include single electron transfer (ET) reactions (e.g., plastocyanin) and substrate oxidation coupled to dioxygen reduction (e.g., laccases) (2). The unique properties of type 1 sites have been recognized since the 1950s, and those properties have been manipulated in a great many ways (3). In addition to its striking blue color, type 1 Cu centers display E°′ values that are generally higher than for Cu in water (E°′ = 0.16 V), ranging from 0.18 V (stellacyanin) to almost 0.8 V for fungal laccase (4), and ≥1 V estimated for ceruloplasmin (5). This large range of E°′ is even more remarkable given the structural similarities of the proteins and the ligand sets (two His, one Cys, and a more weakly bound Met; Fig. 1). Mutations to Cu-ligating residues have major effects on spectroscopic and redox properties (6, 7), but we now know that exquisite tuning of E°′ is made possible by differences in the surrounding (outer-sphere) amino acids, mainly in terms of hydrophobicity and polarity (8). For example, introduction of several Phe groups around azurin-Cu, without changing the native Cu-ligands, can increase E°′ by almost 100 mV (9). Lu and coworkers (10) previously demonstrated that E°′ (Cu) in azurin could be tuned between∼0mV and 640 mVwith mutations to only three residues. The relative contributions of eachmutation also were delineated in that work. Importantly, mutation to the weakly coordinating Met121 (Met121Leu and Met121Gln) induced ±50-mV changes to E°′, respectively. Those changes are modest, but other mutations to the outer-sphere residue Asn47 and Phe114 contribute additive changes to E°′, enabling tuning over a 600-mV range. This feat was impressive, but a modified protein with E°′ approaching 1 V was still out of reach. In PNAS, Hosseinzadeh et al. (1) extend the above work to produce a modified azurin with E°′ = 0.97 V, which is the highest potential ever reported for azurin-Cu, and among the highest reported for any type 1 Cu site. A site with such a high E°′ value was produced by the addition of only two nature-inspired (Fig. 1) outer-sphere mutations (Met44Phe + Gly116Phe) toAsn47Ser/Phe114Asn/Met121Leu azurin (10). These researchers call this new protein high-potential azurin (HPAz). The mutations strongly affect the reduction potential of the embedded Cu site and have relatively small effects on other characteristic type I spectroscopic signatures (i.e., the protein’s vivid blue color and EPR properties). The ability to tune the reduction potential