Role of Interhelical H-Bonds (WR14-TR67 and W15-S72) in the Hemoglobin Allosteric Reaction Path Evaluated by UV Resonance Raman Spectroscopy of Site-Mutants Daojing Wang, † Xiaojie Zhao, † Tong-Jian Shen, ‡ Chien Ho, ‡ and Thomas G. Spiro* ,† Contribution from the Department of Chemistry, Princeton UniVersity, Princeton, New Jersey 08544, and Department of Biological Sciences, Carnegie Mellon UniVersity, 4400 Fifth AVenue, Pittsburgh, PennsylVania 15213 ReceiVed June 28, 1999. ReVised Manuscript ReceiVed September 8, 1999 Abstract: Hemoglobin residues ThrR67 and Ser72 have been mutated to Val and Ala, respectively, to test the hypothesis that tertiary H-bonds involving these residues play a key role in the allosteric reaction path between the R and the T state. The H-bonds are donated by the indole side chains of TrpR14 and Trp15; they bridge the outer A helices to the inner E helices, which line the distal side of the heme pocket. The mutants fold properly (CD measurements) and form native-like T state contacts, as revealed by UVRR (RR ) resonance Raman) difference spectra between deoxyHb and HbCO, and by the Fe-N (histidine) stretching band in the visible RR spectra of deoxyHb. However, the UVRR intensity of tryptophan bands is diminished in the mutants. This is the expected effect of H-bond elimination, because H-bonding shifts the tryptophan excitation profiles to longer wavelengths, raising the intensity at 229 nm, the wavelength employed in this study. Consistent with this interpretation, the intensity loss for the W3 band is found exclusively at 1558 cm -1 , the position of TrpR14 and Trp15, and not at 1548 cm -1 , the position of the interfacial residue Trp37. The intensity loss is greater for TR67V than for S72A, consistent with crystallographic data showing a shorter N‚‚‚O distance for the H-bond from TrpR14 than from Trp15. The H-bond augmentation of the W3 intensity is calculated to be almost a factor of 2 greater for the former than the latter. UVRR difference spectra obtained 150 ns after photolysis of HbCO reveal negative Tyr and Trp bands for the mutants which are similar to those obtained for native Hb, and are attributed to the first protein intermediate on the allosteric reaction path, R deoxy . However, the Trp intensity loss is diminished for the mutants, supporting the hypothesis that the R deoxy Trp signals arise from weakening of the TrpR14 and Trp15 H-bonds, as a result of increased separation between the A and E helices. This separation is proposed to result from rotation of the EF “clamshell” resulting from F helix displacement away from the heme plane, due to the Fe displacement upon deligation, and E helix motion toward the heme plane as the ligand departs the heme pocket. Introduction While the end states of the allosteric transition in hemoglobin (Hb) are the well-established R and T structures, 1 the reaction path has not been defined and remains a subject of great interest. As part of a continuing program to trace this path with the aid of time-resolved vibrational spectroscopy, 2-6 we have examined the effects of mutating residues ThrR67 and Ser72, which are involved in key tertiary H-bonds. These mutants were designed to test a specific hypothesis 3,4,6 about the first protein motion detectable by ultraviolet resonance Raman (UVRR) spectros- copy, when Hb is induced to undergo the R to T transition by flash photolysis of the CO adduct, HbCO. This motion is proposed to involve displacement of the helices, E and F, which sandwich the heme prosthetic group (Figure 1). These displace- ments would weaken H-bonds connecting the E helix to the A helix, on one hand, and the F helix with the H helix on the other. In our model of the allosteric reaction coordinate, 4,6 these H-bonds are subsequently reformed by following motions of the A and H helices, which serve to move the N and C termini into positions from which they establish the intersubunit salt- bridges that stabilize the T structure. 8 UVRR spectroscopy can monitor the E-A and F-H helix separations, because the interhelical H-bonds involve aromatic residues. The H-bonds connecting the F and H helices are from the penultimate tyrosine residues, R140 and 145 to the main chain carbonyl groups of valine residues R93 and 98, while the H-bonds connecting the E and A helices are from the tryptophan residues R14 and 15 to the hydroxyl side chains of ThrR67 and Ser72 (Figure 1). When tyrosine or tryptophan are H-bond donors, the UVRR excitation profiles are red- shifted, 2 reflecting stabilization of the resonant excited states. As a result, their UVRR intensity is increased for excitation at 229 nm, the wavelength used in our studies. Thus, weakening * To whom correspondence should be addressed. † Princeton University. ‡ Carnegie Mellon University. (1) Perutz, M. Mechanism of CooperatiVity and Allosteric Regulation in Proteins; Cambridge University Press: Cambridge, 1990. (2) Rodgers, K. R.; Su, C.; Subramaniam, S.; Spiro, T. G. J. Am. Chem. Soc. 1992, 114, 3697. (3) Rodgers, K. R.; Spiro, T. G. Science 1994, 265, 1697. (4) Jayaraman, V.; Rodgers, K. R.; Mukerji, I.; Spiro, T. G. Science 1995, 269, 1843. (5) Hu, X.; Frei, H.; Spiro, T. G. Biochemistry 1996, 35, 13001. (6) Hu, X.; Rodgers, K. R.; Mukerji, I.; Spiro, T. G. Biochemistry 1999, 38, 3462. (7) Baldwin, J. M. J. Mol. Biol. 1980, 136, 103. (8) Perutz, M. F. Nature 1970, 228, 726. 11197 J. Am. Chem. Soc. 1999, 121, 11197-11203 10.1021/ja992228w CCC: $18.00 © 1999 American Chemical Society Published on Web 11/19/1999