Dynamics of Allostery in Hemoglobin: Roles of the Penultimate Tyrosine H bonds Janina Kneipp 1 , Gurusamy Balakrishnan 1 , Ruopian Chen 1 Tong-Jian Shen 2 , Sarata C. Sahu 2 , Nancy T. Ho 2 , Janel L. Giovannelli 2 Virgil Simplaceanu 2 , Chien Ho 2 and Thomas G. Spiro 1 * 1 Department of Chemistry Princeton University, Princeton NJ 08544, USA 2 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA15213, USA The tyrosine residues adjacent to the C termini of the hemoglobin (Hb) subunits, aY140 and bY145, are expected to play important structural roles, because the C termini are the loci of T-state quaternary salt-bridges, and because the tyrosine side-chains bridge the H and F helices via H bonds to the aV93 and bV98 carbonyl groups. These roles have been investigated via measurements of oxygen binding, 1 H NMR spectra, resonance Raman (RR) spectra, and time-resolved resonance Raman (TR 3 ) spectra on site mutants in which the H/F H bonds are eliminated by replacing the tyrosine residues with phenylalanine. The TR 3 spectra confirm the hypothesis, based on TR 3 studies of wild-type Hb, that the H/F H bonds break and then re-form during the sub-microsecond phase of the R–T quaternary transition. The TR 3 spectra support the inference from other mutational studies that the ab dimers act as single dynamic units in this early phase, motions of the E and F helices being coupled tightly across the dimer interface. Formation of T quaternary contacts occurs at about the same rate in the mutants as in HbA. However, these contacts are weakened substantially by the Y/F substitutions. Equilibrium perturbations are apparent also, especially for the a-subunits, in which relaxation of the Fe– His bond, strengthening of the A/E interhelical H bond, and weakening of the “switch” quaternary contact in deoxyHb are all apparent. Structural effects are less marked for the b-chain Y/F replacement, but the Bohr effect is reduced by 25%, indicating that the salt-bridge and H bond interactions of the adjacent C terminus are loosened. The a-chain replacement reduces the Bohr effect much more, consistent with the global perturbations detected by the structure probes. q 2005 Elsevier Ltd. All rights reserved. Keywords: hemoglobin; site mutants; tyrosine H bonds; allostery; protein dynamics *Corresponding author Introduction As part of a continuing program to elucidate the mechanism of allostery in hemoglobin (Hb), 1,2 we have replaced the penultimate tyrosine residues in the a and b-chains (aY140 and bY145) with phenylalanine. The tyrosine side-chains donate H bonds to the main-chain carbonyl groups of aV93 and bV98, thereby linking the H and F helices of each Hb subunit (Figure 1(a)). 3 These H bonds, along with a pair of tryptophan H bonds linking the A and E helices, have been suggested to break during the initial protein motion following ligand dissociation. 4 The evidence supporting this pro- posal is the generation of a UV resonance Raman (UVRR) difference spectrum containing negative tyrosine and tryptophan bands, a spectrum labeled R deoxy . 4 Assignment of the tryptophan band was confirmed by site-mutation of the H bond acceptor residues, 2,5 and the present study confirms the assignment of the tyrosine band. The R deoxy spectrum is so named because it is seen also for Hb constructs that have one or more 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Abbreviations used: Hb, hemoglobin; RR, resonance Raman; UVRR, resonance Raman spectroscopy with UV excitation; rHb, recombinant Hb. E-mail address of the corresponding author: spiro@princeton.edu doi:10.1016/j.jmb.2005.11.006 J. Mol. Biol. (2006) 356, 335–353