Catching catalysis in the act: using single crystal KINETICS TO TRAP METHYLAMINE DEHYDROGENASE REACTION INTERMEDIATES FOR STRUCTURAL STUDIES.

Arwen R. Pearson, Teresa De la Mora Rey, Kevin T. Watts, Ed Hoeffner and Carrie M. Wilmot

University of Minnesota, Department of Biochemistry, Molecular Biology & Biophysics, Minneapolis, MN 55455, U.S.A. (pears079@umn.edu)

Methylamine dehydrogenase (MADH) is an a₂b₂ heterotetramer containing a novel quinone cofactor, TTQ, derived from two modified tryptophan residues. It is expressed in response to methylamine, allowing certain bacteria to utilise methylamine as their sole carbon source. MADH catalyses the conversion of methylamine to formaldehyde and ammonia, leaving the TTQ cofactor in a 2e- reduced state. To complete the catalytic cycle, MADH is reoxidised via two successive electron transfer (ET) events.

In the case of the Paracoccus denitrificans enzyme (PD-MADH) the physiologic ET chain involves the protein redox partners amicyanin (a blue-copper protein) and cytochrome c551i. Stable binary (PD-MADH/amicyanin) and ternary (PD-MADH/amicyanin/cytochrome c551i) catalytically competent complexes can be formed and crystallized, and their structures have been solved to better than 2.0Ǻ resolution in the laboratory of F. Scott Mathews (Washington University Medical School, St. Louis).

MADH (TTQ), amicyanin (Cu) and cytochrome c551i (Fe) have spectral features in the visible region that change during catalytic turnover, thus defining spectrally distinct intermediates that reflect the electron distribution in the complex.

Through a novel combination of single crystal visible microspectrophotometry, X-ray crystallography and freeze trapping, reaction intermediates of MADH in complex with the physiological redox partners in the crystalline state have been trapped.

This poster will present the methods used to monitor and trap reaction intermediates in the crystalline state, as well as some preliminary X-ray structural data.