News and Publications

Metalloenzyme Engineering

D.B. Goodin, D.E. McRee, R.A. Musah, J. Hirst, S.K. Wilcox, Y. Cao, R.J. Rosenfeld, G.M. Jensen,* S.W. Bunte,** T.M. Loehr,*** A.E. McDermott,**** F.A. Armstrong*****

* Nexstar Pharmaceuticals, San Dimas, CA
** U.S. Army Research Laboratory, Aberdeen, MD
*** Oregon Graduate Institute, Portland, OR
**** Columbia University, New York, NY
***** Oxford University, Oxford, England

One of the greatest challenges in the design of protein-based catalysts is the simultaneous introduction of specificity and catalysis into the structure. Our research focuses on the structural reengineering of enzymes to develop novel catalysts that can oxidize specific substrates. Our experimental approach involves the integrated use of multidisciplinary techniques, including molecular modeling, protein engineering, x-ray crystallography, electron paramagnetic resonance, electrochemistry, calorimetry, and kinetics. The designed catalysts will have practical usefulness as biosensors and as reagents for stereospecific chemical transformations and bioremediation. More generally, these efforts will aid research in molecular recognition and drug design.

During the past several years, we have achieved a detailed structural and biophysical understanding of a class of oxidative heme enzymes typified by the peroxidases and monooxygenases. More recently, we worked out methods for introducing molecular recognition for small molecules at several sites within such enzymes. These sites can be positioned relative to the chemical environment of the active site to enable one of several types of oxidative catalysis, including delivery of activated oxygen to alkenes, promotion of electron transfer by transition-state stabilization, and new electron-transfer pathways from redox-active metal centers.

Modeling, engineering, and x-ray crystallography were used to construct and structurally characterize 7 examples in which an artificial cavity was created at various positions surrounding the active-site heme center. Each of these cavities binds molecules with specificities that are determined by the complementation of molecular shape and biophysical properties of the surrounding protein structure. Advances in the past year include using cyclic voltammetry to obtain reduction potentials of the ferryl heme of cytochrome c peroxidase mutants, thereby establishing an active-site mutant that contains an electron-transfer center that is intrinsically more reactive than the native enzyme. In addition, resonance Raman spectroscopy was used to characterize the details of metal coordination for a ligand-deficient mutant. In a related study, the first detection of deuterium signals from a paramagnetic protein by solid-state nuclear magnetic resonance established that small-molecule ligands in one of our artificial cavities are well ordered and do not undergo significant dynamic motion on the nuclear magnetic resonance time scale.

Electrostatic potential calculations with a discrete solvent model provided a quantitative determination of how the protein structure controls the energetics for the stabilization of an intrinsic radical species. This study has established the degree to which an engineered cavity at this site can stabilize a developing charge during the transition state for electron transfer. A separate study, combining the use of crystallography, electrostatic calculations, and titration calorimetry, provided a detailed picture of how weak hydrogen bonds contribute to molecular specificity in ligand binding to such cavities.

A significant accomplishment in the past year came from 3 successful instances in which novel substrate oxidation was introduced into a heme peroxidase by cavity complementation; in each case, a different mechanism was used. First, oxidation of small heterocyclic aminothiazoles bound to a buried engineered cavity was due to stabilization of the electrostatic transition state specific for the local environment of the cavity. The similarity of the mechanism to the one used by the native enzyme provides a form of substrate oxidation by chemical rescue. Second, a cavity introduced on the distal heme face bound styrene derivatives that were positioned near the active site to promote stereospecific epoxidation. These reactions are analogous to those promoted by P₄₅₀ enzymes. Finally, a designed metal-ion binding site on the surface of the enzyme that uses both engineered residues and a heme propionate to coordinate redox-active metal ions oxidized manganese by a mechanism similar to that of manganese-dependent ligninase. These examples illustrate the successful development of catalysts that used the native propensity of a reactive cofactor to act on a substrate that is positioned by engineered molecular recognition.

PUBLICATIONS

Cao, Y., Musah, R.A., Wilcox, S.K., Goodin, D.B., McRee, D.E. Protein conformer selection by ligand binding observed with crystallography. Protein Sci., in press.

Jensen, G.M., Bunte, S.W., Warshel, A., Goodin, D.B. Energetics of cation radical formation at the active site tryptophan of cytochrome-c peroxidase and ascorbate peroxidase. J. Phys. Chem., in press.

Liu, K., Williams, J., Lee, H.R., Fitzgerald, M.M., Jensen, G.M., Goodin, D.B., McDermott, A.E. Solid state deuterium NMR of 2-methylimidazole in CcP (H175G): A rigid untethered imidazole. J. Am. Chem. Soc., in press.

Mondal, M.S., Goodin, D.B., Armstrong, F.A. Simultaneous voltammetric comparisons of reduction potentials, reactivities, and stabilities of the high-potential catalytic states of wild-type and distal pocket mutant (W51F) yeast cytochrome-c peroxidase. J. Am. Chem. Soc., in press.

Musah, R.A., Goodin, D.B. Introduction of novel substrate oxidation into a heme peroxidase by cavity complementation: Oxidation of 2-aminothiazole and covalent modification of the enzyme. Biochemistry 36:11665, 1997.

Musah, R.A., Jensen, G.M., Rosenfeld, R.J., Bunte, S.W., McRee D.E., Goodin, D.B. Variation in strength of a CH to O hydrogen bond in an artificial cavity. J. Am. Chem. Soc. 119:9083, 1997.

Sarma, S., DiGate, R.J., Goodin, D.B., Miller, C.J., Guiles, R.D. Effect of axial ligand plane reorientation on electronic and electrochemical properties observed in site directed mutants of rat cytochrome b₅. Biochemistry 36:5658, 1997.

Sun, J., Fitzgerald, M.M., Goodin, D.B., Loehr, T.M. The solution and crystal structures of the H175G mutant of cytochrome-c peroxidase: A resonance Raman study. J. Am. Chem. Soc. 119:2064, 1997.