Scientific Report 2005

Molecular Biology

Metalloenzyme Engineering

D.B. Goodin, C.D. Stout, A.-M.A. Hays, S. Vetter, E.C. Glazer, A.E. Pond, H.B. Gray,* J.R. Winkler,* J.H. Dawson,** T.L. Poulos,*** M.A. Marletta****

* California Institute of Technology, Pasadena, California
** University of South Carolina, Columbia, South Carolina
*** University of California, Irvine, California
**** University of California, Berkeley, California

Our overall goals are to understand the fundamental structural features of metalloenzyme catalysts and to create catalysts for useful chemical reactions. We use a number of techniques in structural biology and spectroscopy and strategies of rational protein redesign and molecular evolution. In the past year, we made progress in several areas.

One area of recent interest has been the design and use of molecular wires as probes for the active sites of enzymes such as cytochrome P450 and nitric oxide synthase (NOS). In an ongoing collaboration with H.B. Gray, California Institute of Technology, we are investigating the binding of these wires, which are specifically designed substrate analogs linked to photochemical or redox-active sensitizers, to the active site of metalloproteins. The wires are being developed to serve as reporters of the active-site environment and as tools to allow rapid deposition or withdrawal of electrons to drive redox catalysis. In addition, luminescent wires that are quenched upon either binding or release from the protein may be useful as imaging agents or as tools for identifying novel enzyme inhibitors.

P450s make up a large family of enzymes responsible for a vast range of biologically important oxidation reactions in mammals, plants, fungi, and bacteria. An important unresolved question concerns how the deeply buried heme cofactor of these enzymes achieves regioselective and stereoselective catalysis of a wide range of substrates. In the past year, we completed a detailed structural analysis by x-ray crystallography of cytochrome P450_cam complexed with 2 sensitizer-linked substrate probes, D4A and D8A. These probes differ in length but bind identically at the substrate end of the wire (Fig. 1).

Fig. 1. Crystal structure at 1.6 Å of P450cam containing D8A, a synthetic molecular wire. The adamantyl substrate analog is observed at the camphor binding site for wires of different lengths. Changes in the F and G helices in response to wire length illustrate the conformational flexibility in these regions that may be responsible for the diversity of substrate recognition by P450s.

Significant changes in the protein structure near the F and G helices accommodate the changes in linker length. These changes are similar to those that may be responsible for substrate-binding specificity of mammalian P450s and indicate that prokaryotic enzymes have similar conformational flexibility. These changes also suggest the nature of the dynamic intermediates that must exist transiently in solution during substrate entry and product egress. The conformational change associated with movement of the F and G helices is transmitted to a backbone carbonyl at the active site of the enzyme, which has been implicated in gating the critical peroxy-bond cleavage that activates the enzyme for catalysis.

In other studies, we are designing and synthesizing specific pterin-based molecular wires for the active site of NOS. NOSs are complex enzymes used for the production of nitric oxide from arginine and play many critical roles in biological signal transduction. As thiolate coordinate heme enzymes, they have structural and functional similarities to P450s. One unique feature is the role played by the pterin cofactor of NOS. Recent results suggest that the pterin donates an electron to either the heme or the substrate at defined steps in the catalytic mechanism. In the past year, we designed and synthesized a series of pterin analogs tethered to sensitizers containing redox-active ruthenium to be used as specific molecular triggers and probes of the NOS active site. In addition, we measured the FeIII/II and FeII/I couples by direct cyclic voltammetry of inducible NOS in organic films on graphite electrodes.

These studies allow easy and rapid measurements of electron transfer between the enzyme and the electrode surface and enabled us to detect the interconversion of several coordination states of the enzyme. These studies, coupled with the use of molecular wires as mediators of electron transfer at electrode surfaces, will provide a new way to prove the function of NOS and related enzymes.

Publications

Hays, A.-M., Dunn, A.R., Chiu, R., Gray, H.B., Stout, C.D. Goodin, D.B. Conformational states of cytochrome P450_cam revealed by trapping of synthetic molecular wires. J. Mol. Biol. 344:455, 2004.

Udit, A.K., Belliston-Bittner, W., Glazer, E.C., Nguyen, Y.H.L., Gillon M., Hill, M.G., Marletta, M.A., Goodin, D.B. Gray, H.B. Redox couples of inducible nitric oxide synthase. J. Am. Chem. Soc. 127:11212, 2005.