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Type 1 copper (T1Cu) centers are crucial in biological electron transfer (ET) processes, exhibiting a wide range of reduction potentials (E°′T1Cu) to match their redox partners and optimize ET rates. While tuning E°′T1Cu in mononuclear T1Cu proteins like azurin has been successful, it is more difficult for multicopper oxidases. Specifically, while replacing axial methionine to leucine in azurin increased its E°′T1Cu by ~100 mV, the corresponding M298L mutation in small laccase from Streptomyces coelicolor (SLAC) unexpectedly decreased its E°′T1Cu by 12 mV. X-ray crystallography revealed two axial water molecules in M298L-SLAC, leading to the decrease of E°′T1Cu due to decreased hydrophobicity. Structural alignment and molecular dynamics simulation indicated a key difference in T1Cu axial loop position, leading to the different outcome upon methionine to leucine mutation. Based on structural analyses, we introduced additional F195L and I200F mutations, leading to partial removal of axial waters, a 122-mV increase in E°′T1Cu, and a 7-fold increase in kcat/KM from M298L-SLAC. These findings highlight the complexity of tuning E°′T1Cu in multicopper oxidases and provide valuable insights into how structure-based protein engineering can contribute to the broader understanding of T1Cu center, E°′T1Cu and reactivity tuning for applications in solar energy transfer, fuel cells, and bioremediation. 

The primary and secondary coordination spheres of metal binding sites in metalloproteins have been investigated extensively, leading to the creation of high-performing functional metalloproteins; however, the impact of the overall structure of the protein scaffold on the unique properties of metalloproteins has rarely been studied. A primary example is the binuclear CuA center, an electron transfer cupredoxin domain of photosynthetic and respiratory complexes and, recently, a protein co-regulated with particulate methane and ammonia monooxygenases. The redox potential, Cu–Cu spectroscopic features, and a valence delocalized state of CuA are difficult to reproduce in synthetic models, and every artificial protein CuA center to-date has used a modified cupredoxin. Here we present a fully functional CuA center designed in a structurally non-homologous protein, cytochrome c peroxidase (CcP), by only two mutations (CuACcP). We demonstrate with UV-visible absorption, resonance Raman, and MCD spectroscopy that CuACcP is valence delocalized. CW and pulsed (HYSCORE) X-band EPR show it has a highly compact gz area and small Az hyperfine principal value with g and A tensors that resemble axially perturbed CuA. Stopped-flow kinetics found that CuA formation proceeds through a single T2Cu intermediate. The reduction potential of CuACcP is comparable to native CuA and can transfer electrons to a physiological redox partner. We built a structural model of the designed Cu binding site from EXAFS and validated it by mutation of coordinating Cys and His residues, revealing that a triad of residues (R48C, W51C, and His52) rigidly arranged on one α-helix is responsible for chelating the first Cu atom and that His175 stabilizes the binuclear complex by rearrangement of the CcP heme-coordinating helix. This design is a demonstration that a highly conserved protein fold is not uniquely necessary to induce certain characteristic physical and chemical properties to a metal redox center. 

Abstract The key to type 1 copper (T1Cu) function lies in the fine tuning of the Cu^II/Ireduction potential (E°′_T1Cu) to match those of its redox partners, enabling efficient electron transfer in a wide range of biological systems. While the secondary coordination sphere (SCS) effects have been used to tuneE°′_T1Cuin azurin over a wide range, these principles are yet to be generalized to other T1Cu‐containing proteins to tune catalytic properties. To this end, we have examined the effects of Y229F, V290N and S292F mutations around the T1Cu of small laccase (SLAC) fromStreptomyces coelicolorto match the highE°′_T1Cuof fungal laccases. Using ultraviolet‐visible absorption and electron paramagnetic resonance spectroscopies, together with X‐ray crystallography and redox titrations, we have probed the influence of SCS mutations on the T1Cu and correspondingE°′_T1Cu. While minimal and smallE°′_T1Cuincreases are observed in Y229F‐ and S292F‐SLAC, the V290N mutant exhibits a majorE°′_T1Cuincrease. Moreover, the influence of these mutations onE°′_T1Cuis additive, culminating in a triple mutant Y229F/V290N/S292F‐SLAC with the highestE°′_T1Cuof 556 mV vs. SHE reported to date. Further activity assays indicate that all mutants retain oxygen reduction reaction activity, and display improved catalytic efficiencies (k_cat/K_M) relative to WT‐SLAC.