Role of interhelical H-bonds (W alpha 14-T alpha 67 and W beta 15-S beta 72) in the hemoglobin allosteric reaction path evaluated by UV resonance raman spectroscopy of site-mutants

Hemoglobin residues Thr alpha 67 and Ser beta 72 have been mutated to Val a nd Ala, respectively, to test the hypothesis that tertiary H-bonds involvin g these residues play a key role in the allosteric reaction path between th e R and the T state. The H-bonds are donated by the indole side chains of T rp alpha 14, and Trp beta 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 reveal ed by UVRR (RR = resonance Raman) difference spectra between deoxyHb and Kb CO, and by the Fe-N (histidine) stretching band in the visible RR spectra o f 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, ra ising the intensity; at 229 nm, the wavelength employed in this study. Cons istent with this interpretation, the intensity loss for the W3 band is foun d exclusively at 1558 cm(-1), the position of Trp alpha 14 and Trp beta 15, and not at 1548 cm(-1), the position of the interfacial residue Trp beta 3 7: The intensity loss is greater for T alpha 67V than for S beta 72A, consi stent with crystallographic data showing a shorter N ... O distance for the H-bond from Trp alpha 14 than from Trp beta 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 photo lysis of HbCO reveal negative Tyr and Trp bands for the mutants which are s imilar to those obtained for native Hb, and are attributed to the first pro tein intermediate on the allosteric reaction path, R-deoxy. However, the Tr p intensity loss is diminished for the mutants, supporting the hypothesis t hat the R-deoxy Trp signals arise from weakening of the Trp alpha 14 and Tr p beta 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 "cla mshell" resulting from F helix displacement away from the heme plane, due t o the Fe displacement upon deligation, and E helix motion toward the heme p lane as the ligand departs the heme pocket.