The long-term aim of the molecular biophysics research in the Warncke Laboratory is to understand fundamental principles governing catalysis in biological systems. In particular, our focus is on radical-mediated catalysis in metalloenzymes. We target the investigation of radical reactions in the coenzyme B12 (adenosylcobalamin) dependent enzymes. This system offers distinct advantages for the detailed biophysical study of the radical pair separation, hydrogen atom transfer and radical rearrangement reactions that are integral to radical catalysis in many enzymes. The work is funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the NIH, and is part of the recent surge of experimental and theoretical interest in B12 systems. We address factors that influence radical reactivity by using spectroscopic techniques, in combination with biochemical approaches. Our principal spectroscopic technique is pulsed-electron paramagnetic resonance (EPR), which is performed on a home-constructed wideband spectrometer. We also use absorption spectroscopy. Our past work has focused on the determination of structures of cryo-trapped biradical intermedates. In parallel with these continuing studies, we are now developing time-resolved methods, which probe radical pair reaction dynamics by using magnetic resonance and optical techniques following pulsed-laser photolysis of the B12 molecule. The biochemical work, which involves preparation of the enzyme, enzymological studies, and manipulation of samples for spectroscopy, is vital for discovering novel signals and controlling the reactions for concise spectroscopic inquiry.   Some representative results are presented in short form in the links below. For more details, view our publications.

PHOTOCATALYSIS in BIOLOGICAL and ARTIFICIAL SYSTEMS

DIRECT DETECTION OF PERNICIOUS BIOLOGICAL FREE RADICALS

Natural systems have evolved that use light to perform chemical reactions with remarkable quantum yield and reasonable conservation of photon energy. For instance, the enzyme, DNA photolyase, uses a long wavelength (low energy) UV photon to reverse thymine dimerization caused by short wavelength (high energy) UV damage of DNA. This reaction proceeds with near unit quantum yield. Another well-known example is the bacterial photosynthetic reaction center protein, which performs a photon-triggered sequence of electron transfer reactions that result in a 22 Å electron-hole separation in 200 ps. We are interested in determining the molecular factors that govern efficient photon-to-reaction product conversion in these systems. Static and time-resolved techniques of pulsed-EPR and optical spectroscopy are being used to probe the reaction mechanisms in the DNA photolyase and RC protein systems. This information will be used to design natural and artificial catalysts to perform desired reactions.

Free radical damage is a biomedical problem of broad scope. Free radicals have been shown or proposed to be involved in a wide range of maladies, including xenobiotic chemical toxicity, atherosclerosis, rheumatoid arthritus, cancer. Aging itself has been proposed to be caused by radical damage accumulated over the lifetime of the organism. However, the relatively small amounts of free radicals and their transient existence (high reactivity) have impeded efforts to draw clear connections between radical generation, presence and cell/tissue damage. Spin trap molecules were developed to form stable adducts with the radicals, thus allowing EPR detection. The spin trap methods have contributed key insights into the presence of radicals. Nevertheless, the presence of the spin traps truncates radical damage cascades, and the formation of the spin-adduct can obscure the identity of the original radical. Using our ability to sensitively detect unpaired spins on the microsecond to seconds timescale, we are developing parallel biochemical and pulsed-EPR spectroscopic strategies to directly detect radical intermediates and demonstrate causative links between their presence and damage.