Electronic structure and reactivity of resting and intermediate forms of the tetranuclear copper sulfur active site of nitrous oxide reductase [electronic resource]
Keywords: Chemistry.

This dissertation focuses on studies of the enzyme nitrous oxide reductase (N2OR), which catalyzes the last step of the nitrogen cycle, the two electron reduction of N2O to dinitrogen and water. While nitrous oxide reductase has been studied since its discovery in 1982, several questions remain about the mechanism of N2O reduction by its tetranuclear copper sulfide active site (CuZ*, a Cu4S cluster). The recent discovery of a different structural form of the active site that contains a second inorganic sulfur edge ligand (CuZ, a Cu4S2 cluster) raises questions about which form of the site is responsible for the catalytic reduction of N2O. The identification of an intermediate CuZo form of the cluster in single turnover of fully reduced CuZ* with N2O provides the opportunity to gain insight into the geometric and electronic structure of an intermediate in catalysis, leading to new insight into the nature of N2O reduction and electron transfer in N2OR. In this dissertation, we use spectroscopic methods correlated to density functional theory (DFT) calculations to examine the electronic structure of resting and intermediate forms of the tetranuclear copper active site of nitrous oxide reductase. These are correlated to reactivity studies on the resting and intermediate forms, leading to insight into the mechanism of N2O reduction. N2OR has been found to have two structural forms of its tetranuclear copper active site, the Cu4S CuZ* form and the 4Cu2S CuZ form. In Chapter 2, EPR, resonance Raman, and MCD spectroscopies have been used to determine the accessible redox states of these sites, showing that the CuZ* site accesses the 1-hole and fully reduced redox states while the CuZ site accesses the 2-hole and 1-hole redox states. Single turnover reactions of N2OR for CuZ and CuZ* poised in these redox states and steady state turnover assays with different proportions of CuZ and CuZ* show that only fully reduced CuZ* is catalytically competent in rapid turnover with N2O. An intermediate 1-hole form of the tetranuclear copper active site of N2OR has been observed in single turnover of fully reduced N2OR (containing CuZ*) with a stoichiometric amount of N2O. In Chapter 3, a combination of spectroscopic methods and density functional theory (DFT) calculations are used to determine the geometric and electronic structure of the CuZo intermediate. Electron paramagnetic resonance (EPR), absorption, and magnetic circular dichroism (MCD) spectroscopies show that there is a redistribution of spin density in the CuZo intermediate from mostly localized on CuI in resting CuZ* to more evenly delocalized over CuI and CuIV. Two Cu-S vibrations are observed in the resonance Raman spectrum at 425 and 413 cm-1, the latter showing a -3 cm-1 O18 solvent isotope shift. Two possible structural models for the CuZo intermediate are evaluated using DFT calculations and the intermediate is shown to have a terminal hydroxide ligand coordinated to CuIV and stabilized by a hydrogen bond to a nearby lysine residue. Insights into the reactivity of N2OR that arise from the structure of the CuZo intermediate are discussed. Marcus theory is used to show that the rapid reduction of CuZo in turnover is due to a 6.4 kcal/mol higher driving force for the reduction of the metastable CuZo intermediate relative to resting CuZ*. Terminal hydroxide coordination in the CuZo intermediate yields insight into the nature of N2O binding and reduction, suggesting a mechanism in which N2O coordinates in a [mu] -1,3 fashion to the fully reduced site, with hydrogen bonding from the second sphere lysine, and two electrons are transferred to N2O via a single Cu center to accomplish N-O bond cleavage. Spectroscopic and computational methods have been used to determine the protonation state of the edge sulfur ligand in the Cu4S2 CuZ form of the active site of N2OR in its 1-hole and 2-hole redox states. 1-hole CuZ is shown to have a [mu] 2-thiolate edge ligand from the observation of S-H bending modes in the resonance Raman spectrum at 450 and 492 cm-1 that have significant deuterium isotope shifts (-137 cm-1) and are not perturbed up to pH 10. Models of the 1-hole and 2-hole CuZ sites are evaluated using DFT calculations and correlated to the spectral features of the two redox states. The energy of deprotonation of a [mu] 2-thiolate ligand in 2-hole CuZ relative to 1-hole CuZ is used to show that 2-hole CuZ has a [mu] 2-sulfide edge ligand at neutral pH. The slow two electron reduction of N2O by 1-hole CuZ, which involves donation of both an electron and a proton, is discussed and a possible route from 2-hole CuZ to CuZ* via reaction with O2 is suggested.