Redox processes are at the heart of numerous functions in chemistry and biology, from long-range electron transfer (ET) in photosynthesis and respiration to catalysis in industrial and fuel cell research. Nature accomplishes these functions by employing only a limited number of redox-active agents. A long-standing issue in these fields is how redox potentials are fine-tuned over a broad range with little change to the redox-active site or ET properties. Resolving this issue will not only advance our fundamental understanding of the roles of long-range, non-covalent interactions in redox processes, but also allow for design of redox-active proteins having tailor-made redox potentials for applications such as artificial photosynthetic centers1,2 or fuel cell catalysts3 for energy conversion. We have shown here that two important secondary coordination sphere interactions, hydrophobicity and hydrogen-bonding, are capable of tuning the reduction potential of the cupredoxin azurin (Az) over a 700 mV range, surpassing the highest and lowest reduction potentials reported for any mononuclear cupredoxin, without perturbing the metal binding site beyond what is typical for the cupredoxin family of proteins. We also demonstrate that the effects of individual structural features are additive and that redox potential tuning of Az is now predictable across the full range of cupredoxin potentials.

Magnetotactic bacteria have evolved complex subcellular machinery to construct linear chains of magnetite nanocrystals that allow the host cell to sense direction. Each mixed-valent iron nanoparticle is mineralized from soluble iron within a membrane-encapsulated vesicle termed the magnetosome, which serves as a specialized compartment that regulates the iron, redox, and pH environment of the growing mineral. To dissect the biological components that control this process, we have carried out a genetic and biochemical study of proteins proposed to function in iron mineralization. In this study, we show that the redox sites of c-type cytochromes of the Magnetospirillum magneticum AMB-1 magnetosome island, MamP and MamT, are essential to their physiological function and that ablation of one or both heme motifs leads to loss of function, suggesting that their ability to carry out redox chemistry in vivo is important. We also develop a method to heterologously express fully heme-loaded MamP from AMB-1 for in vitro biochemical studies, which show that its Fe(III)-Fe(II) redox couple is set at an unusual potential (−89 ± 11 mV) compared with other related cytochromes involved in iron reduction or oxidation. Despite its low reduction potential, it remains competent to oxidize Fe(II) to Fe(III) and mineralize iron to produce mixed-valent iron oxides. Finally, in vitro mineralization experiments suggest that Mms mineral-templating peptides from AMB-1 can modulate the iron redox chemistry of MamP.

Evolutionary links between type 1 blue copper (T1 Cu), type 2 red copper (T2 Cu), and purple Cu(A) cupredoxins have been proposed, but the structural features and mechanism responsible for such links as well as for assembly of Cu(A) sites in vivo are poorly understood, even though recent evidence demonstrated that the Cu(II) oxidation state plays an important role in this process. In this study, we examined the kinetics of Cu(II) incorporation into the Cu(A) site of a biosynthetic Cu(A) model, Cu(A) azurin (Cu(A)Az) and found that both T1 Cu and T2 Cu intermediates form on the path to final Cu(A) reconstitution in a pH-dependent manner, with slower kinetics and greater accumulation of the intermediates as the pH is raised from 5.0 to 7.0. While these results are similar to those observed previously in the native Cu(A) center of nitrous oxide reductase, the faster kinetics of copper incorporation into Cu(A)Az allowed us to use lower copper equivalents to reveal a new pathway of copper incorporation, including a novel intermediate that has not been reported in cupredoxins before, with intense electronic absorption maxima at ~410 and 760 nm. We discovered that this new intermediate underwent reduction to Cu(I), and proposed that it is a Cu(II)-dithiolate species. Oxygen-dependence studies demonstrated that the T1 Cu species only formed in the presence of molecular oxygen, suggesting the T1 Cu intermediate is a one-electron oxidation product of a Cu(I) species. By studying Cu(A)Az variants where the Cys and His ligands are mutated, we have identified the T2 Cu intermediate as a capture complex with Cys116 and the T1 Cu intermediate as a complex with Cys112 and His120. These results led to a unified mechanism of copper incorporation and new insights regarding the evolutionary link between all cupredoxin sites as well as the in vivo assembly of Cu(A) centers.

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Within Cu-containing electron transfer active sites, the role of the axial ligand in type 1 sites is well defined, yet its role in the binuclear mixed-valent Cu A sites is less clear. Recently, the mutation of the axial Met to Leu in a Cu A site engineered into azurin (Cu A Az) was found to have a limited effect on E 0 relative to this mutation in blue copper (BC). Detailed low-temperature absorption and magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance studies on Cu A Az (WT) and its M123X (X = Q, L, H) axial ligand variants indicated stronger axial ligation in M123L/H. Spectroscopically validated density functional theory calculations show that the smaller ΔE 0 is attributed to H 2 O coordination to the Cu center in the M123L mutant in Cu A but not in the equivalent BC variant. The comparable stabilization energy of the oxidized over the reduced state in Cu A and BC (Cu A ∼ 180 mV; BC ∼ 250 mV) indicates that the S(Met) influences E 0 similarly in both. Electron delocalization over two Cu centers in Cu A was found to minimize the Jahn-Teller distortion induced by the axial Met ligand and lower the inner-sphere reorganization energy. The Cu-S(Met) bond in oxidized Cu A is weak (5.2 kcal/ mol) but energetically similar to that of BC, which demonstrates that the protein matrix also serves an entatic role in keeping the Met bound to the active site to tune down E 0 while maintaining a low reorganization energy required for rapid electron transfer under physiological conditions. spectroscopy | reduction potential | energy transduction pathway L ong-range electron transfer (ET) is vital to a wide range of biological processes, including two key energy transduction pathways essential for life: H 2 O oxidation in photosynthesis and O 2 reduction in respiration (1, 2). Nature has adapted a conserved cupredoxin fold motif (i.e., the Greek-key β barrel) to construct two evolutionarily linked, but structurally distinct Cucontaining ET proteins (3-5). These are the mononuclear type 1 (T1) or blue copper (BC) and binuclear purple Cu A proteins. The first coordination sphere of the classic BC sites [e.g., plastocyanin (Pc) and azurin (Az)] consists of a trigonally distorted tetrahedral environment where Cu resides in an equatorial plane formed by one S(Cys) and two N(His) ligands and has an axial S (Met) ligand (Fig. 1A) (Fig. 1B) (8-11). Both sites carry out rapid, efficient longrange ET with rates on the order of 10 3 -10 5 s −1 (12, 13).Although BC proteins use a Cu + /Cu 2+ redox couple, the binuclear Cu A sites use a (Cu 1+ -Cu 1+ )/(Cu 1.5+ -Cu 1.5+ ) redox cycle. The oxidized form of Cu A is mixed-valent (MV), with a highly covalent Cu 2 S 2 core that gives rise to its unique spectroscopic features. The unpaired electron is fully delocalized over the two Cu centers and exhibits a characteristic seven-line 63,65 Cu hyperfine splitting pattern in electron paramagnetic resonance (EPR) spectroscopy (14, 15). Maintaining valence delocalization even in the presence of a low symmetry protein ...