Postdoctoral Scholar, California Institute of Technology
Mechanisms of biological activation of small molecules using first row transition metals
Laccases belong to the family of multicopper oxidases (MCOs) that couple oxidation of a wide spectrum of substrates to the four-electron reduction of dioxygen (O2) to water. Laccases typically contain four copper ion sites arranged in two centers: one type 1 blue copper center and one trinuclear cluster (TNC) consisting of one type 2 copper and a binuclear type 3 copper pair. Dioxygen binding, activation, and reduction are believed to proceed at the TNC with electrons delivered from the type 1 site.
The precise mechanism of coupling O2 reduction to substrate oxidation is a subject of considerable fundamental and technological significance. Of particular interest is the mechanism by which laccases oxidize refractory substrates such as phenols and lignin.
To gain deeper insight into laccase catalysis, I have cloned the gene for a recombinant multicopper oxidase from the hyperthermophilic bacterium Thermus thermophilus HB27 (Tth-MCO), and expressed the enzyme as a 6Histagged protein in Escherichia coli. Several mutants of the recombinant enzyme have been prepared by site–directed mutagenesis and investigated using various structural and spectroscopic techniques.
I will be discussing my recent findings on the reaction pathways and intermediates involved in the enzymatic reaction.
Abstract: Laccases are part of the family of multicopper oxidases (MCOs), which couple the oxidation of substrates to the four electron reduction of O2 to H2O. MCOs contain a minimum of four Cu's divided into Type 1 (T1), Type 2 (T2), and binuclear Type 3 (T3) Cu sites that are distinguished based on unique spectroscopic features. Substrate oxidation occurs near the T1, and electrons are transferred approximately 13 Å through the protein via the Cys-His pathway to the T2/T3 trinuclear copper cluster (TNC), where dioxygen reduction occurs. This review outlines the electron transfer (ET) process in laccases, and the mechanism of O2 reduction as elucidated through spectroscopic, kinetic, and computational data. Marcus theory is used to describe the relevant factors which impact ET rates including the driving force, reorganization energy, and electronic coupling matrix element. Then, the mechanism of O2 reaction is detailed with particular focus on the intermediates formed during the two 2e(-) reduction steps. The first 2e(-) step forms the peroxide intermediate, followed by the second 2e(-) step to form the native intermediate, which has been shown to be the catalytically relevant fully oxidized form of the enzyme.
Pub.: 13 Jan '15, Pinned: 30 Jun '17
Abstract: A synthetic heme-Cu CcO model complex shows selective and highly efficient electrocatalytic 4e(-)/4H(+) O2-reduction to H2O with a large catalytic rate (>10(5) M(-1) s(-1)). While the heme-Cu model (FeCu) shows almost exclusive 4e(-)/4H(+) reduction of O2 to H2O (detected using ring disk electrochemistry and rotating ring disk electrochemistry), when imidazole is bound to the heme (Fe(Im)Cu), this same selective O2-reduction to water occurs only under slow electron fluxes. Surface enhanced resonance Raman spectroscopy coupled to dynamic electrochemistry data suggests the formation of a bridging peroxide intermediate during O2-reduction by both complexes under steady state reaction conditions, indicating that O-O bond heterolysis is likely to be the rate-determining step (RDS) at the mass transfer limited region. The O-O vibrational frequencies at 819 cm(-1) in (16)O2 (759 cm(-1) in (18)O2) for the FeCu complex and at 847 cm(-1) (786 cm(-1)) for the Fe(Im)Cu complex, indicate the formation of side-on and end-on bridging Fe-peroxo-Cu intermediates, respectively, during O2-reduction in an aqueous environment. These data suggest that side-on bridging peroxide intermediates are involved in fast and selective O2-reduction in these synthetic complexes. The greater amount of H2O2 production by the imidazole bound complex under fast electron transfer is due to 1e(-)/1H(+) O2-reduction by the distal Cu where O2 binding to the water bound low spin Fe(II) complex is inhibited.
Pub.: 01 Oct '15, Pinned: 30 Jun '17
Abstract: Our long-time niche in synthetic biological inorganic chemistry has been to design ligands and generate coordination complexes of copper or iron ions or both, those reacting with dioxygen (O2) or nitrogen oxides (e.g., nitric oxide (NO(g)) and nitrite (NO2(-))) or both. As inspiration for this work, we turn to mitochondrial cytochrome c oxidase, which is responsible for dioxygen consumption and is also the predominant target for NO(g) and nitrite within mitochondria. In this Account, we highlight recent advances in studying synthetic heme/Cu complexes in two respects. First, there is the design, synthesis, and characterization of new O2 adducts whose further study will add insights into O2 reductive cleavage chemistry. Second, we describe how related heme/Cu constructs reduce nitrite ion to NO(g) or the reverse, oxidize NO(g) to nitrite. The reactions of nitrogen oxides occur as part of CcO's function, which is intimately tied to cellular O2 balance. We had first discovered that reduced heme/Cu compounds react with O2 giving μ-oxo heme-Fe(III)-O-Cu(II)(L) products; their properties are discussed. The O-atom is derived from dioxygen, and interrogations of these systems led to the construction and characterization of three distinctive classes of heme-peroxo complexes, two high-spin and one low-spin species. Recent investigations include a new approach to the synthesis of low-spin heme-peroxo-Cu complexes, employing a "naked" synthon, where the copper ligand denticity and geometric types can be varied. The result is a collection of such complexes; spectroscopic and structural features (by DFT calculations) are described. Some of these compounds are reactive toward reductants/protons effecting subsequent O-O cleavage. This points to how subtle improvements in ligand environment lead to a desired local structure and resulting optimized reactivity, as known to occur at enzyme active sites. The other sector of research is focused on heme/Cu assemblies mediating the redox interplay between nitrite and NO(g). In the nitrite reductase chemistry, the cupric center serves as a Lewis acid, while the heme is the redox active center providing the electron. The orientation of nitrite in approaching the ferrous heme center and N-atom binding are important. Also, detailed spectroscopic and kinetic studies of the NO(g) oxidase chemistry, in excellent agreement with theoretical calculations, reveal the intermediates and key mechanistic steps. Thus, we suggest that both chemical and biochemical heme/Cu-mediated nitrite reductase and NO(g) oxidase chemistry require N-atom binding to a ferrous heme along with cupric ion O-atom coordination, proceeding via a three-membered O-Fe-N chelate ring transition state. These important mechanistic features of heme/Cu systems interconverting NO(g) and nitrite are discussed for the first time.
Pub.: 06 Aug '15, Pinned: 30 Jun '17