Photodissociation and Rebinding of H 2 O to Ferrous Sperm Whale Myoglobin Don C. Lamb,* ,²,‡ Valeri Prusakov, § Niklas Engler, ² Andreas Ostermann, ² Peter Schellenberg, | Fritz G. Parak, ² and G. Ulrich Nienhaus ‡,| Fakulta ¨ t fu ¨ r Physik E17, Technische UniVersita ¨ t Mu ¨ nchen D-85747 Garching, Germany Department of Physics, UniVersity of Illinois 1110 West Green Street, Urbana, Illinois 61801-3080 Institute of Chemical Physics, Russian Academy of Science, Moscow, Russia Department of Biophysics, UniVersity of Ulm D-89069 Ulm, Germany ReceiVed NoVember 3, 1997 The investigation of the binding of small ligands has provided valuable insights into the relations among structure, dynamics, and function of proteins. 1 In particular, the binding of carbon monoxide (CO) and dioxygen (O 2 ) to myoglobin (Mb) has been studied in great detail and lead to the notion of conformational substates 1a (CS) and protein relaxations. 1b,d,2 The changes in ligand affinity for CO and O 2 imposed by the polypeptide chain prevents the endogenously produced levels of CO from being toxic. 3 Water, while being abundant in the physiological environ- ment and even present in the heme pocket, does not bind to ferrous Mb at physiological temperatures. Here, we provide evidence that water binds to ferrous Mb at cryogenic temperatures in a photodissociable complex. Through reduction of aquometmyo- globin (Mbmet) at 20 K, we succeeded in producing an Fe(II) low-spin configuration at the active center with the water molecule still bound. The water ligand was photodissociated with a short laser pulse, and the nonexponential rebinding kinetics were monitored as a function of time and temperature. Mbmet can be reduced at low temperature using γ-rays, 4 X-rays, 5 visible light, 6 or photochemical methods. 7 Here, we used either X-rays to reduce samples that were optically thick or tris- (2,2′,bipyridine)ruthenium(II) ([Ru(bpy) 3 ] 2+ ) as a photoactivated reducing agent. The metastable intermediate state formed is structurally similar to Mbmet, with the water molecule still bound, but the heme iron is in the Fe(II) low-spin configuration. The absorption spectrum of the intermediate state has been characterized 4b,7 as well as the relaxation of the intermediate state to the equilibrium deoxy Mb structure at temperatures above 160 K. 5b,c,7 Upon illumination of the low-spin intermediate species (Fe II - MbH 2 O) with low intensity light at 20 K, there is a decrease in the amplitude of the Soret band at 23 360 cm -1 and a new band at 22 600 cm -1 appears, indicative of a high-spin-like state (Figure 1a). After the sample was warmed above 60 K and cooled back to 20 K, the new band essentially disappeared while the Soret band of the Fe II MbH 2 O state returned to its original value, implying that this deoxy-like state reverted back to the Fe(II) low- spin intermediate state. Photodissociation of a water ligand from the low-spin iron with concomitant conversion to high-spin was considered as a sensible explanation of the spectral changes. To obtain further support for this scenario, we investigated band III near 13 200 cm -1 which has been assigned to a porphyrin-to- iron a 2u f d yz charge-transfer transition. 8 It is only observed in Fe(II) high-spin, five-coordinate hemes. Because of the ap- proximately 1000-fold weaker absorption of this band compared with the Soret, a concentrated protein solution (17 mM) with a sample thickness of 2 mm was used. It was mounted between thin Mylar windows and irradiated with X-rays for 17.5 h at 80 K. Subsequently, the sample was transferred at low temperature to an optical cryostat mounted within the optical spectrometer. Spectra were taken at 20 K before and after illumination with a 20 mW laser diode at 14 600 cm -1 for 30 min. As shown in Figure 1b, the charge-transfer band III, indicative of a high-spin deligated species, indeed appears with a peak position of 13 000 cm -1 upon illumination. Consequently, we conclude that the ² Technische Universita ¨t Mu ¨nchen. ‡ University of Illinois. § Russian Academy of Science. | University of Ulm. (1) (a) Austin, R. H.; Beeson, K. W.; Eisenstein, L.; Frauenfelder, H.; Gunsalus, I. C. Biochemistry 1975, 14, 5355-5373. (b) Ansari, A.; Berendzen, J.; Bowne, S. F.; Frauenfelder, H.; Iben, I. E. T.; Sauke, T. B.; Shyamsunder, E.; Young, R. D. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 5000-5004. (c) S ˇ rajer, V.; Reinisch, L.; Champion, P. M. J. Am. Chem. Soc. 1988, 110, 6656- 6670. (d) Steinbach, P. J.; Ansari, A.; Berendzen, J.; Braunstein, D.; Chu, K.; Cowen, B. R.; Ehrenstein, D.; Frauenfelder, H.; Johnson, B.; Lamb, D. C.; Luck, S.; Mourant, J. R.; Nienhaus, G. U.; Ormos, P.; Phillip, R.; Xie, A.; Young, R. D. Biochemistry 1991, 30, 3988-4001. (e) Ehrenstein, D.; Nienhaus, G. U. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9681-9685. (f) Tian, W. D.; Sage, J. T.; S ˇ rajer, V.; Champion, P. M. Phys. ReV. Lett. 1992, 68, 408-411. (g) Balasubramanian, S.; Lambright, D. G.; Marden, M. C.; Boxer, S. G. Biochemistry 1993, 32, 2202-2212. (h) Post, F.; Doster, W.; Karvounis, G.; Settles, M. Biophys. J. 1993, 64, 1833-1842. (i) Hagen, S. J.; Hofrichter, J.; Eaton, W. A. Science 1995, 269, 959-962. (j) Johnson, J. B.; Lamb, D. C.; Frauenfelder, H.; Mu ¨ller, J. D.; McMahon, B.; Nienhaus, G. U.; Young, R. D. Biophys. J. 1996, 71, 1563-1573. (k) Ober, C.; Burkardt, M.; Winkler, H.; Trautwein, A. X.; Zharikov, A. A.; Fischer, S. F. Parak, F.; Eur. Biophys. J. 1997, 26, 227-237. (2) Agmon, N.; Hopfield, J. J. J. Chem. Phys. 1983, 79, 2042-2053. (3) Springer, B. A.; Sligar, S. G.; Olson, J. S.; Phillips, G. N., Jr. Chem. ReV. 1994, 94, 699-714. (4) (a) Magonov, S. N.; Davydov, R. M.; Blumenfeld, L. A.; Vilu, R. O.; Arutjunjan, A. M.; Sharonov, Yu. A. Molek. Biol. 1978, 12, 947-956 (in Russian). (b) Magonov, S. N.; Davydov, R. M.; Blumenfeld, L. A.; Vilu, R. O.; Arutjunjan, A. M.; Sharonov, Yu. A. Molek. Biol. 1978, 12, 1182-1189 (in Russian). (c) Blumenfeld, L. A. Physics of bioenergetic processes; Springer Series Synergetics; Springer-Verlag: Berlin, Heidelberg, New York, Tokyo 1983; Vol. 16. (d) Blumenfeld, L. A.; Burbajev, D. S.; Davydov, R. M. The Fluctuating Enzyme; Welch, G. R., Ed.; John Wiley & Sons: New York, 1986; pp 369-402. (e) Prusakov, V. E.; Stukan, R. A.; Parak, F. Khim. Fiz. 1990, 9, 44-50 (in Russian). (5) (a) Parak, F.; Prusakov, V. E. Hyperfine Interact. 1994, 91, 885-890. (b) Prusakov, V. E.; Steyer, J.; Parak, F. Biophys. J. 1995, 68, 2524-2530. (c) Prusakov, V. E.; Parak, F.; Chekunaev, N. I.; Goldanskii, V. I. Biophysics 1996, 5, 1005-1015. (6) Gu, Y.; Li, P.; Sage, J. T.; Champion, P. M. J. Am. Chem. Soc. 1993, 115, 4993-5004. (7) Lamb, D. C.; Ostermann, A.; Prusakov, V.; Parak, F. G. Eur. Biophys. J. 1998, 27, 113-125. (8) Eaton, W. A.; Hofrichter, J. Methods Enzymol. 1981, 76, 175-261. Figure 1. (a) The Soret spectrum of the intermediate state at 20 K before and after illumination with a laser diode. Absorption from [Ru(bpy)3] 2+ has been subtracted to more clearly demonstrate the changes in the Soret region. The sperm whale Mb concentration was 5 mM. (b) The difference spectrum from before and after illumination measured in the near-IR using a 17 mM sperm whale Mb sample. 2981 J. Am. Chem. Soc. 1998, 120, 2981-2982 S0002-7863(97)03781-5 CCC: $15.00 © 1998 American Chemical Society Published on Web 03/13/1998