Study of Water Binding to Low-Spin Fe(III) in Cytochrome P450 by Pulsed ENDOR and Four-Pulse ESEEM Spectroscopies D. Goldfarb,* ,²,‡ M. Bernardo, § H. Thomann,* ,§ P. M. H. Kroneck, ⊥ and V. Ullrich ⊥ Contribution from the Department of Chemical Physics, The Weizmann Institute of Science, RehoVot 76100, Israel, Exxon Research and Engineering Co., Route 22 East, Annandale, New Jersey 08801, and Faculty of Biology, UniVersity of Konstanz, UniVersita ˚ tstrasse 10, D-78434 Konstanz, Germany ReceiVed April 24, 1995 X Abstract: Cytochrome P450cam (CP450cam) was studied by pulsed ENDOR and two- and four-pulse ESEEM spectroscopies. Spectra were recorded and simulated at the three principal g-values of the rhombic EPR spectrum. The four-pulse ESEEM experiment gave a direct measure of the anisotropic hyperfine interaction for the protons. Using the point dipole approximation this gives a Fe-H distance of 2.6 Å. The measured anisotropic hyperfine interaction reduced the number of hyperfine interaction parameters required to simulate the ENDOR line shapes. Both the four-pulse ESEEM frequencies and the ENDOR spectra at all three principal g-values could be satisfactorily simulated using two magnetically equivalent protons and a water orientation similar to that obtained in our previous 17 O ESEEM study. Thus, the pulsed ENDOR and four-pulse ESEEM results are self-consistent with the 17 O ESEEM data and indicate that the axial ligand is a water molecule rather than an OH - ligand. The isotropic hyperfine value derived from the numerical simulations is in agreement with previous values derived from proton NMR relaxation studies. Introduction Cytochrome P450 (CP450) enzymes are ubiquitous O 2 activators consisting of a protoporphyrin IX prosthetic group as the active site. 1-4 The best characterized CP450 enzyme is the CP450 camphor from Pseudomonas Putida(CP450cam) for which several high-resolution crystal structures were reported. 5-9 In the substrate free CP450cam enzyme the Fe 3+ is low spin and hexacoordinated. 5 Upon camphor binding the Fe 3+ becomes high-spin and pentacoordinated. 6,7,10 A cysteine thiolate sulfur constitutes the proximal ligand which remains bound to the metal upon substrate binding. From X-ray diffraction studies it has been concluded that the distal ligand, which is removed upon camphor binding, is either a water molecule or a hydroxide ion, with a Fe-O distance of 2.28 Å. 5 The possible mechanisms relating the substrate binding, the coordination number, the spin state equilibrium, and the redox potential where discussed by Poulos and Raag. 9,11 These authors also argued that OH - is the more likely sixth axial ligand. Room temperature NMR relaxation measurements on substrate free P450cam showed a significant enhancement of the water proton relaxation 12 indicating that one or more strongly exchangeable protons, with an isotropic hyperfine constant of 2.2-3.1 MHz, are within 2.6-2.9 Å from the heme iron. 13 Electron nuclear double resonance (ENDOR) measurements also showed the existence of strongly coupled exchangeable proton(s) with hyperfine splittings that are in agreement with the NMR results. 14 The possibility that the signal is due to proton(s) bound to the cysteinyl sulfur could not, however, be ruled out. 14 In a recent communication the two broad ENDOR peaks corresponding to proton couplings of 10 and 15.2 MHz were attributed to H 2 O signals in two major conformational substates of the H 2 O cluster in the distal heme pocket. 15 Recently, the binding characteristics of the distal ligand in substrate free CP450cam were measured using 17 O electron spin-echo envelope modulation (ESEEM) spectroscopy of the enzyme after exchange with H 2 17 O. 16 From the ESEEM results the 17 O hyperfine and nuclear quadrupole interaction parameters were determined. Based on the magnitude and orientation of the quadrupole interaction it was concluded that at pH 7.5, the distal ligand is a water molecule rather than OH - . The water molecule was found to be oriented with the H-O-H bisector parallel to the g z direction which is approximately perpendicular * Authors to whom correspondence may be addressed. ² Work by performed while on sabbatical leave at Exxon from 8/92 to 8/93. ‡ The Weizmann Institute of Science. § Exxon Research and Engineering Co. ⊥ University of Konstanz. X Abstract published in AdVance ACS Abstracts, March 1, 1996. (1) (a) Sato, R.; Omura, T. Cytochrome P-450; Academic Press: New York, 1979. (b) Hayaishi, O. Molecular Mechanism of Oxygen ActiVation; Academic Press: New York, 1994. (2) White, R. E.; Coon, M. J. Annu. ReV. Biochem. 1980, 49, 315. (3) Coon, M. J.; White, R. E. Dioxygen Binding and ActiVation by Metal Centers; Spiro, T., Ed.; John Wiley and Sons: New York, 1980; pp 73- 123. (4) Murray, R. I.; Fisher, M. T.; Debrunner, P. G.; Sligar, S. G. Metalloproteins, Harrison, P. M., Ed.; Verlag Chemie: Florida, Basel 1985; Vol. I, pp 157-206. (5) Poulos, T. L.; Finzel, B. C.; Howard, A. J. Biochemistry 1986, 25, 5314. (6) Poulos, T. L.; Finzel, B. C.; Gunsalus, I. C.; Wagner, C. W.; Karaut, J. J. Biol. Chem. 1985, 260, 16122. (7) Poulos, T. L.; Finzel, B. C.; Howard, A. J. Mol. Biol. 1987, 195, 687. (8) Raag, R.; Poulos, T. L. Biochemistry 1989, 28, 7586. (9) Raag, R.; Poulos, T. L. Biochemistry 1989, 28, 917. (10) Tsai, R.; Yu, C. A.; Gunsalus, I. C.; Peisach, J.; Blumberg, W.; Orme-Johnson, W. H.; Beinert, H. Proc. Natl. Acad. Sci. U.S.A. 1970, 66, 1157. (11) Poulos, T. L.; Raag, R. FASEB J. 1992, 6, 674. (12) Griffin, B. W.; Peterson, J. A. J. Biol. Chem. 1975, 250, 6445. (13) Philson, S. B.; Debrunner, P. G.; Schmidt, P. G.; Gunsalus, I. C. J. Biol. Chem. 1979, 254, 10173. (14) LoBrutto, R.; Scholes, C. P.; Wagner, G. C.; Gunsalus, I. C.; Debrunner, P. G. J. Am. Chem. Soc. 1980, 102, 1167. (15) Fann, Y.-C.; Gerber, N. C.; Osmulski, P. A.; Hager, L. P.; Sligar, S. G.; Hoffman, B. M. J. Am. Chem. Soc. 1994, 116, 5989. (16) Thomann, H.; Bernardo, M.; Goldfarb, D.; Kroneck, P. M. H.; Ullrich, V. J. Am. Chem. Soc. In press. 2686 J. Am. Chem. Soc. 1996, 118, 2686-2693 0002-7863/96/1518-2686$12.00/0 © 1996 American Chemical Society