This project focuses on the structures of transient oxygen intermediates occurring in the catalytic cycles of iron enzymes. Specific focus is on the resolution of atomic vibrations of oxygenic metal ligands and their protonation states using continuous flow resonance Raman spectroscopy. Using a unique, state of the art experimental setup, designed and implemented by the PI, we will examine isotope-difference Raman spectra of species that are formed for a short time following the start of the reactions. The major advantage of our approach is the ability to carry out reactions in liquid samples at temperatures as low as -70?C with minuscule dead volume. This allows us to slow down the rates of chemical reactions by orders of magnitude and permits close examination of the early phases of the reaction without the need for prohibitive amounts of biological samples. We will examine two classes of iron enzymes. First, we will study transient intermediates that we recently identified in the mono-nuclear non-heme iron and ?-ketoglutarate dependent dioxygenase, TauD. We will characterize these intermediates at various temperatures using excitations across the UV/visible spectrum in order to isolate their spectral signatures. These species will be further investigated using isotope-labeled substrates and medium. A range of synthetic modeling, computational and comparative studies will be carried out that will allow unambiguous identification of the structures of the new species. The second group includes two key bacterial di-iron enzymes. Methane monooxygenase is a powerful analog of cytochrome P450 in higher organisms, which is capable of oxidizing methane to methanol. Its highly oxidized intermediate Q contains an unprecedented FeIVFeIV core. Ribonucleotide reductase plays a key role in DNA replication and is a good model for the corresponding mammalian enzyme. Its highly oxidized intermediate X is responsible for activation of the enzyme by generating a protein radical. Structures of both X and Q have been studied extensively over the last two decades, but remain controversial. Using our novel approach we seek to identify the number, type, and protonation states of oxygenic ligands in X and Q, which is critical for resolving their specific catalytic mechanisms.
Short-lived highly oxidized oxygen-metal complexes are the catalytic centerpieces of many enzymes exhibiting a diverse range of critical physiological functions. Using a unique experimental setup, which us allows to dramatically slow down chemical reactions, we seek to establish the detailed structures of such intermediates. We will investigate three representative enzymes: ribonucleotide reductase is involved in DNA replication and two bacterial oxygenases provide important models of analogous enzymes in mammals.