Binding and Electron Transfer between Putidaredoxin and Cytochrome P450cam. Theory and Experiments Adrian E. Roitberg,* Marcia J. Holden, Martin P. Mayhew, Igor V. Kurnikov, † David N. Beratan, † and Vincent L. Vilker Contribution from the Biotechnology DiVision, National Institute of Standards and Technology, Building 222, Room A353, Gaithersburg, MD 20899 ReceiVed NoVember 24, 1997 Abstract: We present a detailed atomic level view of the interactions between cytochrome P450cam (CYP101) and its natural redox partner, putidaredoxin (Pdx). A combined theoretical (Poisson-Boltzmann electrostatic calculations, electron transfer pathways, and molecular dynamics) and experimental (site-directed mutagenesis and kinetic analysis) study is used to pinpoint surface residues in both proteins that are important for electron transfer, binding, or both. We find a situation where the electrostatically complementary regions at the surface of both proteins overlap strongly with regions that have large electron transfer couplings to the redox centers. This means that a small surface patch in each protein is involved in binding and electron transfer. A dominant electron transfer pathway is identified, corresponding to an electron leaving the reduced Fe 2 S 2 in Pdx, going through Cys39 and Asp38, and transferring across the interprotein interface to Arg112 (CYP101), then to a heme propionate group, and finally to the heme iron center. Introduction A necessary step in understanding biological function in multiple metalloprotein redox systems is the description of protein-protein binding and electron transfer between two distant metal centers. In this paper, we present experimental and theoretical analyses of the residues that are involved in the binding and the electron transfer pathways of the cytochrome P450cam (CYP101) and putidaredoxin (Pdx) protein complex. The in vivo function of the CYP101 enzymatic complex is to catalyze the stereoselective oxidation of camphor to 5-exo- hydroxycamphor. The bacterium Pseudomonas putida, from which this enzyme was originally extracted and purified, can survive by using camphor as its sole carbon and energy source. 1 The full catalytic cycle of CYP101 starts with NADH, which reduces the FAD-containing protein putidaredoxin reductase (PdR). PdR then transfers an electron to putidaredoxin (Pdx). Two distinct electron transfer steps are needed from Pdx to CYP101 in order to enable catalysis. The first electron transfer step can be attained by a number of compounds, but the second one can only be done in the presence of Pdx. The origin of this difference in behavior is yet to be resolved, and in the present paper, we will not distinguish between these two steps. It is however important to note that there is probably a single binding site associated with each of the independent electron transfer events. 2,3 Of the three proteins in this cycle, only the structure of CYP101 (MW 45 000) has been elucidated using X-ray methods. 4,5 A solution structure of Pdx (MW 11 594) has been elucidated using NMR methods 6 and deposited in the PDB 7 database under the code 1PUT. It is worth noting that the NMR experiments on this protein are hampered by the presence of the paramagnetic Fe 2 S 2 cluster. This fact has the effect of largely displacing and grossly widening the NMR lines for most nuclei lying within an 8 Å sphere of the cluster. The route followed by Pochapsky consisted of collecting the largest possible number of NOE restraints and dihedral restraints and a number of paramagnetic broadening restraints. There are also data to support the idea that the regions close to the metallic cluster are homologous among a series of ferredoxins. The large homology between Pdx and the ferredoxins of Spirulinas platensis and Anabaena at the cluster region enabled Pochapsky to model the active site region. The mix of homology section and the actual NMR data were then minimized with the usual methodologies. 6 The stability of that structure when immersed in a box of water with proper inclusion of electrostatic terms has been recently studied, 8 and it was found that the structure proposed by Porchapsky holds very well under long molecular dynamics conditions. Very recently, an X-ray structure of a highly homologous protein has been solved; 9 a truncated bovine adrenodoxin was elucidated at a resolution of 1.85 Å and has a main-chain RMSD deviation to Pdx of only 1.64 Å. Recently, Pochapsky et al. 10 proposed a model for the structure of the Pdx-CYP101 complex based on molecular dynamics simula- tions that attempted to place the iron-sulfur center of Pdx as * To whom correspondence should be addressed. E-mail: adrian@nist.gov. † Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260. (1) Ortiz de Montellano, P. R. Cytochrome P450: structure, mechanism, and biochemistry, 2nd ed.; Plenum Press: New York,1995. (2) Hintz, M. J.; Mock, D. M.; Peterson, L. L.; Tuttle, K.; Peterson, J. A. J. Biol. Chem. 1982, 257, 14324. (3) Unno, M.; Shimada, H.; Toba, Y.; Makino, R.; Ishimura, Y. J. Biol. Chem. 1996, 271, 17869-17874. (4) Poulos, T. L.; Finzel, B. C.; Howard, A. J. J. Mol. Biol. 1987, 195, 687. (5) Poulos, T. L.; Raag, R. FASEB J. 1992, 6, 674-679. (6) Pochapsky, T. C.; Ye, X. M.; Ratnaswamy, G.; Lyons, T. A. Biochemistry 1994, 33, 6424-6432. (7) Bernstein, F. C.; Koetzle, T. F.; Williams, G. J. B.; Meyer, J. E. F.; Brice, M. D.; Rodgers, J. R.; Kennard, O.; Shimanouchi, T.; Tasumi, M. J. Mol. Biol. 1977, 112, 535-542. (8) Roitberg, A. E. Biophys. J. 1997, 73, 2138-2148. (9) Muller, A.; Muller, J. J.; Muller, Y. A.; Uhlmann, H.; Bernhardt, R.; Heinemann, U. Structure 1998, 6, 269-280. 8927 J. Am. Chem. Soc. 1998, 120, 8927-8932 S0002-7863(97)03990-5 CCC: $15.00 © 1998 American Chemical Society Published on Web 08/21/1998