Stanford Advisors

2017-18 Courses

All Publications

  • A Binuclear CuA Center Designed in an All α-Helical Protein Scaffold. Journal of the American Chemical Society Mirts, E. N., Dikanov, S. A., Jose, A. n., Solomon, E. I., Lu, Y. n. 2020; 142 (32): 13779–94


    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 coregulated 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 nonhomologous protein, cytochrome c peroxidase (CcP), by only two mutations (CuACcP). We demonstrate with UV-visible absorption, resonance Raman, and magnetic circular dichroism spectroscopy that CuACcP is valence delocalized. Continuous wave and pulsed (HYSCORE) X-band EPR show it has a highly compact g z area and small A z 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 extended X-ray absorption fine structure spectroscopy 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(II) 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 in a metal redox center.

    View details for DOI 10.1021/jacs.0c04226

    View details for PubMedID 32662996

  • Chloride Control of the Mechanism of Human Serum Ceruloplasmin (Cp) Catalysis. Journal of the American Chemical Society Tian, S., Jones, S. M., Jose, A., Solomon, E. I. 2019


    Unraveling the mechanism of ceruloplasmin (Cp) is fundamentally important toward understanding the pathogenesis of metal-mediated diseases and metal neurotoxicity. Here we report that Cl-, the most abundant anion in blood plasma, is a key component of Cp catalysis. Based on detailed spectroscopic analyses, Cl- preferentially interacts with the partially reduced trinuclear Cu cluster (TNC) in Cp under physiological conditions and shifts the electron equilibrium distribution among the two redox active type 1 (T1) Cu sites and the TNC. This shift in potential enables the intramolecular electron transfer (IET) from the T1 Cu to the native intermediate (NI) and accelerates the IET from the T1 Cu to the TNC, resulting in faster turnover in Cp catalysis.

    View details for DOI 10.1021/jacs.9b03661

    View details for PubMedID 31203609

  • Geometric and Electronic Structure Contributions to O-O Cleavage and the Resultant Intermediate Generated in Heme-Copper Oxidases. Journal of the American Chemical Society Schaefer, A. W., Roveda, A. C., Jose, A. n., Solomon, E. I. 2019; 141 (25): 10068–81


    This study investigates the mechanism of O-O bond cleavage in heme-copper oxidase (HCO) enzymes, combining experimental and computational insights from enzyme intermediates and synthetic models. It is determined that HCOs undergo a proton-initiated O-O cleavage mechanism where a single water molecule in the active site enables proton transfer (PT) from the cross-linked tyrosine to a peroxo ligand bridging the heme FeIII and CuII, and multiple H-bonding interactions lower the tyrosine p Ka. Due to sterics within the active site, the proton must either transfer initially to the O(Fe) (a high-energy intermediate), or from another residue over a ∼10 Å distance to reach the O(Cu) atom directly. While the distance between the H+ donor (Tyr) and acceptor (O(Cu)) results in a barrier to PT, this separation is critical for the low barrier to O-O cleavage as it enhances backbonding from Fe into the O22- σ* orbital. Thus, PT from Tyr precedes O-O elongation and is rate-limiting, consistent with available kinetic data. The electron transfers from tyrosinate after the barrier via a superexchange pathway provided by the cross-link, generating intermediate PM. PM is evaluated using available experimental data. The geometric structure contains an FeIV═O that is H-bonded to the CuII-OH. The electronic structure is a singlet, where the FeIV and CuII are antiferromagnetically coupled through the H-bond between the oxo(Fe) and hydroxo(Cu) ligands, while the CuII and Tyr• are ferromagnetically coupled due their delocalization into orthogonal magnetic orbitals on the cross-linked His residue. These findings provide critical insights into the mechanism of efficient O2 reduction in HCOs, and the nature of the PM intermediate that couples this reaction to proton pumping.

    View details for DOI 10.1021/jacs.9b04271

    View details for PubMedID 31146528