This award in the Inorganic, Bioinorganic and Organometallic Chemistry program supports Professor Brian R. Crane at Cornell University to study long-range electron transfer between cytochrome c peroxidase (CcP) and cytochrome c (Cc) to ascertain how molecular associations, conformational dynamics, and hole hopping control long-range electron transfer rates. Substitution of heme cofactors by Zn-porphyrins will enable photo-activation of redox chemistry. Spectroscopic rate measurements are to be carried out in a set of crystalline complexes between CcP and Cc that display a range of association states and reactivities, set redox center environment and fix molecular orientation. X-ray diffraction studies will directly correlate structure with reactivity. Through mutagenesis, small-molecule complementation, cross-linking, temperature variation, and solvent substitution studies, parameters affecting hopping reactions and protein/solvent dynamics will be systematically probed, all in the context of single crystals. Experiments will be guided and reconciled by computational studies that assess electronic coupling between donor and acceptor sites as a function of conformational state and solvent structure.
The proposed work will reveal how specific parameters, such as bridge structure, conformation, redox potentials and hopping site environment affect long-range ET in heterogeneous systems similar to those synthetically accessible for device development. Dissemination of findings will be facilitated by submission of data to public databases and the development of new web-based tools that readily allow the visualization of structure/function relationships for electron donor/acceptor interactions. The proposed research will train undergraduate and graduate students in bio-, physical- and inorganic chemistry and interface well with the investigators formal teaching efforts in these areas.
This project explored the role of long-range multi-step electron transfer (ET) in metalloenzyme catalysis. Proteins utilize aromatic residues (primarily Trp and Tyr) and organic cofactors in so-called "hole-hopping" reactions to accelerate and control electron flux into enzyme active centers. To probe key chemical parameters in these processes we studied the electron transfer partners cytochrome c peroxidase (CcP) and cytochrome c (CcP) and nitric oxide synthase (NOS). CcP:Cc ET involves hole-hopping through a conserved Trp residue, wherease NOS relies on cofactor-mediated hole-hopping for oxidation activation reactions. Our previous work had shown that different association modes of the CcP:Cc complex possessed surprisingly similar ET reactivities. Our new computational studies on these systems rationalized this activity and established the importance of hole-hopping through Trp191 in accelerating interfacial ET between CcP and Cc. We then substituted Trp191 with Phe, Tyr and Gly residues and showed that only Tyr could support both peroxidase activity and rapid photoinduced ET in a mimetic system wherein the CcP heme is replaced by the photosensitizer Zn-porphyrin. Remarkably Tyr-substituted CcP forms a Cpd I state similar to that of the Trp-containing protein. We also were able to complement the Trp191Gly variant with a set of small molecules that bind in the same position as the Trp indole; however, none were active. We believe inactivity of compounds was largely due to their rapid exchange rates with solvent; thereby underscoring the importance of structural stability in a hopping site. Crystal structures were determined of all complexes to establish the association states of the partners and active site conformations. Computational studies on the CcP:Cc system also indicated that ET operates in the Marcus inverted region. We verified this assertion by testing the driving force dependence of photoinduced ET. Variants of Cc that perturbed the heme redox potential indeed showed that ET slows with increasing driving force. These studies provide one of the few experimental measures of reorganization energy for interprotein ET and demonstrate that optimization of driving force will not necessarily optimize rates of reactivity. Furthermore, changes to solvation (through altered molecular associations) can tune ET reactivity by modulating reorganization energies. These results also have implications for design of soft material electronics and the electronic behavior of interfacial systems. Our work on NOS demonstrates how localized charge movement controls enzymatic catalysis, both in the confines of the active center and in the delivery of charges to the active center. We confirm the role of the cofactor hopping site in facilitating the second step of the reaction by what amounts to electrocatalysis. One-electron redox chemistry of the cofactor within the protein is decidedly different than the two-electron reactivity seen in solution. We have also established that the two stages of NOS catalysis involve different reactive heme-oxygen species, whose formation and stability is controlled by substrate. Importantly, a hydroperxo, rather than a peroxo intermediates is the key reactant in the second stage. This distinguishes the mechanism from a nucleophilic heme-oxygen reactions as seen in P-450 aromatase. These results have implications for understanding oxidation activation by metalloenzymes and the design of catalysts for oxidation reactions of organic substrates. Finally, to produce our metalloproteins of study we developed a ferrochelatase co-expression method that considerably produces yields of recombinant protein. We have shared this method with over 20 academic laboratories and two companies.
