John L. McCracken, Ph.D. - US grants

The long-range objectives of the research proposed in this application are to characterize the ligation structures of nickel in two important classes of nickel enzymes and to further develop the pulsed-EPR techniques of electron spin-echo envelope modulation (ESEEM) and electron spin-echo detected ENDOR spectroscopy for the characterization of paramagnetic centers in biological systems. The specific experiments aimed at achieving the first goal involve the study of Ni(III) and Ni(I) model complexes of biologically relevant structure using cw-EPR, cw-ENDOR, ESEEM, and ESE- ENDOR spectroscopies. Ni(III) peptide model complexes will be studied using ESEEM, cw- and ESE-ENDOR methods to obtain magnetic coupling parameters and coupling tensor orientations for 14N, 2H, and 1H containing ligands to determine how these magnetic parameters are related to structure. These data will provide the necessary background for interpreting the results of published and ongoing ESE and ENDOR investigations of the nickel center in Ni-Fe hydrogenases. Ni(I) derivatives of cofactor F430, thought to play a key role in methane production by methanogenic bacteria, will also be studied using pulsed- and cw-EPR techniques. Here, ligand hyperfine coupling constants and tensor orientations will yield detailed information concerning the axial bonding properties of the cofactor and the consequences of cofactor reduction as related to changes in corphin macrocycle distortion and "puckering". These properties are thought to be key elements in defining the unique chemistry of F430 and its catalytic function in methyl reductases. During the course of this research, ESE-ENDOR methodology will be developed and its capabilities compared directly with those of cw-ENDOR. In addition, current methods of data analysis will be improved. Because metalloenzymes serve as catalysts for a number of essential oxidation-reduction reactions in biological systems with paramagnetic ions being involved at some stage in these processes, the results of this research will provide pertinent information and enhanced spectroscopic tools for the investigation of many health-related problems.

Abstract 9402016 Dye This research involves the synthesis and characterization of crystalline salts and vapor-deposited thin films that contain alkali metal anions (alkalides) or trapped electrons (electrides) and cations that are complexed by cyclic or bicyclic poly-oxa molecules. Particular emphasis will be given to the study of the optical, magnetic and electrical properties of single crystals using a variety of techniques. Because different electrides have widely different properties, and since their behavior often is dependent on the thermal history of the sample, new complexants will be synthesized to provide a wider assortment of crystalline electrides. Recent theoretical treatments have pointed out the importance of anionic cavities and empty channels in electrides and the extended nature of the unpaired electron wavefunction in molecular electrides. To relate experiment to theory, simple complexants for Li+, such as 9-crown-3, 12-crown-3 and cryptand 1.1.1 will be synthesized and used to prepare electrides for study. The use of alkalides and electrides to prepare nanoscale transition metal and intermetallic particles, both free and in zeolite cages will be studied. %%% This research is concerned with the synthesis and characterization of crystalline salts and vapor-deposited thin films that contain alkali metal anions (alkalides) or trapped electrons (electrides) and cations. Alkalides and electrides are two new, interesting classes of materials. Electrides, in particular, provide the unique opportunity to examine the behavior of electrons that do not "belong" to a particular atom or molecule, but rather, occupy the holes and channels between close-packed large complexed cations. They provide the opportunity to study such phenomena as non-metal to metal transitions in systems that approach a lattice of nearly free electrons, and magnetic interactions and electronic conductivity in 1, 2, and 3 dimensions. Emphasis will be given to the study of the optical, magnetic and ele ctrical properties of single crystals using many characterization methods.

DESCRIPTION (provided by applicant): The long range goals of this research project are to understand the role played by paramagnetic centers in the catalytic mechanism of several enzymes, to develop correlations between the detailed electronic structures, gained using advanced Electron Paramagnetic Resonance (EPR) methods, and chemical reactivity, and to further the development of advanced EPR methods and data analysis strategies for solving problems in biological systems. The enzymes to be studied are two multicopper oxidases, FET3p and R. vernicifera laccase, and two alpha-ketoglutarate dependent non-heme iron dioxygenases, taurine dioxygenase and AIkB. All of these proteins catalyze reactions that involve paramagnetic intermediate states at some point in their mechanistic cycle. The experiments proposed in this grant will use EPR spectroscopy at both 9 and 95 GHz. Conventional, continuous wave studies will be used along with the "advanced" EPR techniques of cw- and spin echo detected-ENDOR, and ESEEM in 1- and 2-dimensions. These advanced technologies can be used at both conventional, X-band frequencies (9 GHz) and at W-band (95 GHz). The specific aims of the proposal focus on characterizing the detailed electronic structures of the metal ions that form the catalytic sites of the metalloenzymes under study. Rapid Freeze Quench methods will be used to trap key catalytic intermediates. The data will provide information on the electronic structure of these metals, the coordination of ligands and how both electronic structure and ligand bonding change during catalytic turnover. EPR methods are uniquely suited to obtain this information for paramagnetic centers. Of the four enzymes to be studied, two are directly tied to health related issues. The AIkB protein has recently been identified as having the ability to catalyze the repair of methylated adenine and cytosine bases in both single- and double-stranded DNA. The Fet3p enzyme is a multicopper oxidase that shows ferroxidase activity and thus, shares a common reactivity with ceruloplasrnin, a mammalian rnulticopper ferroxidase that is vital to metal ion homeostasis.

The long-range goal of the spectroscopic studies described in this core project are to use the structural information gained from X-ray crystallographic studies of Prostaglandin H Synthase (PGHS) and Cytochrome c Oxidase (CcOX) as platforms for exploring the details of their catalytic mechanisms, and to develop new spectroscopic methods for the study of metal centers in biological systems. Vibrational spectroscopies, Resonance Raman and FTIR, which provide detailed information regarding bond strengths, will be extended to energy regimes where metal-ligand bonding can be better characterized When combined with small-volume, stop-flow kinetic techniques, these optical methods provide a unique pathway for characterizing catalytic intermediates. The advanced EPR methods that will be utilized in this core project, allow one to measure to wave function that house an unpaired electron spin and gain an understanding of the electronic structure of a catalytic site. The EPR development work that will be undertaken in this core project includes the development of a pulse-microwave/pulse-magnetic field method for improving our capability to resolve hyperfine couplings for high-spin paramagnetic metal centers, the application of several new coherence transfer pulsed EPR methods for the measurement of protein radical conformation and ligand hyperfine couplings, and the parallel development of analysis strategies to support these measurements. Because the development work described in this proposal is driven by specific questions being addressed for CcOX and PGHS, the methodologies defined during the course of this project will be generally useful to the biomedical community.

DESCRIPTION: The long range objectives of the research proposed in this application are to understand the catalytic mechanisms of several enzymes by using advanced electron paramagnetic resonance (EPR) spectroscopic methods to probe the detailed electronic structure of their catalytic sites and to gain an understanding of how these systems control chemical reactivity. The enzymes to be studied are copper-containing amine oxidases, methylamine dehydrogenases, methylmalonyl Coenzyme A Mutase, and carbonmonoxide dehydrogenase. All of these proteins catalyze reactions that involve paramagnetic intermediate states at some point during their cycle. This work will involve further development and use of pulsed and double resonance EPR experiments to measure magnetic and electronic interactions between the unpaired electron spin of the transient paramagnetic species and the magnetic nuclei that comprise their environment. Such hyperfine couplings provide a direct measurement of the highest occupied molecular orbital of the paramagnetic center and thus yield information on the valence electron distribution that is involved in catalysis. Such information is difficult or impossible to obtain by other spectroscopic methods and by X-ray crystallography. Such information will be correlated with the results of kinetic and other structural measurements. In particular the results will be related to electron transferring oxidation/reduction processes.

DESCRIPTION: (Adapted from the applicant's abstract) Free radicals in biological millieu generally provoke destructive chemistry, owing to their reactivity and non-specific chemistry. Recently, however, a new class of enzymes has been identified in which reactive radical species are generated from amino-acid or modified amino-acid side chains during the normal course of catalysis. These paramagnetie species appear to be integral to the proper function of these enzymes. Some of the principles by which these radicals operate are emerging and can be summarized as follows: a) the redox-active side-chain usually occurs in close proximity to a metal center that is involved in generating the radical species; b) once formed, the radical functions in catalysis by abstracting hydrogen-atoms from substrate; and c) the role of the protein includes stabilizing the reactive radical species so as to prevent non-specific chemistry. Understanding the means by which these principles are implemented at the molecular level is a long-term objective of the project. To do this, the applicants intend to focus on the 02-evolving (Mn)4YZ Center in Photosysytem II, which has kinetic properties that allow them to trap specific intermediates in the catalytic process, and on galactose oxidase, for which redox-linked ligand exchange processes and substitution-induced spin-density modulation have been proposed. A principal aim of the project is to develop and generalize low-frequency FTIR difference techniques so that direct observation of metal-substrate ligand, metal cofactor, and cofactor vibrational modes becomes possible. Employing this methodology will require instrument development in combination with directed mutagenesis, isotope-substitution, inorganic model compound, electrochemical, and computational studies. They intend to continue their magnetic-resonance work on the radical-enzyme class by developing methods to treat radical-pairs in hyperfine interaction with nuclei in their immediate vicinity and by refining their understanding of the factors that control spin density distributions in the radical enzyme class.

This proposal requests funds for the purchase of a Bruker E-680 94 GHz/ 9GHz continuous-wave (cw) and pulsed Electron Paramagnetic Resonance spectrometer for use by a team of six NIH-supported investigators at Michigan State University. This instrument would provide new experimental capabilities to the major user group that would be very beneficial to their ongoing R-01 and P-01 funded research programs. The 94 GHz, or W-band, capability would allow us to address problems in metallobiochemistry that are not feasible with our current X- band (9 GHz) spectrometers. The ability to carry out W-band EPR studies in pulsed-mode may alleviate several of the problems anticipated with high frequency studies such as difficulties associated with using magnetic field modulation to detect broad absorption lines. For site- directed spin labeling (SDSL) studies, the W-band spectrometer will supply data required to obtain accurate distances between nitroxides in doubly-labeled proteins. Pulsed EPR experiments at both X- and W-band will provide more detailed information for these SDSL studies that will lead to more precise structural information. Cw and pulsed ENDOR methods will be available at both microwave frequencies. The advantages of performing ENDOR spectroscopy at high field have been pioneered by the Hoffman lab at Northwestern over the past 15 years and include enhanced sensitivity for studies of nuclei with small magnetic moments, improved spectral resolution and more precise structural information that stems from orientation-selective studies. The pulsed EPR capability at X-band will provide the major users with two- dimensional EPR/ESEEM and time domain detection techniques that will benefit several of the proposed studies. The instrument has a flexible pulse programmer, a sophisticated data acquisition system and a complement of sample probes that make it an excellent platform for the development of biological applications. Purchase of an E-680 spectrometer will shift our development efforts away from hardware building and towards solving biomedical problems.

With this award from the Chemistry Research Instrumentation and Facilities: Multi-user (CRIF:MU) program, Professor John McCracken and colleagues Gary Blanchard, Robert Ofoli, Greg Swain and David Weliky from Michigan State University will acquire a series of components to build a rapid acquisition, picosecond fluorescence lifetime and anisotropy imaging system. The award will enhance research training and education at all levels, especially in areas of study such as (a) fluorescence lifetime and anisotropy imaging of lipid structures, (b) optical and electrochemical characterization of biomimetic nanostructured interfaces, (c) chemically modified electrodes for studies of graphene and thin carbon films, and (d) imaging of membrane perturbations induced by viral fusion peptides and proteins.

A rapid acquisition, picosecond fluorescence lifetime and anisotropy imaging system is important to the study of a broad range of structurally heterogeneous and fluid interfaces such as those in biological membranes. These experiments provide novel information on molecular motion and energy transfer, and specifically how these properties vary with the physical condition and chemical composition of the interface. This instrumentation will support not only research activities but also research training to graduate and undergraduate students at Michigan State University and nearby institutions such as Saginaw Valley State University. The instrument will also support international interactions with the University of Warsaw, University of Bath and Shaanxi Normal University as well as activities with local high school students.