High quality ultraviolet resonance Raman (UVRR) spectra with 230-nm excitation are reported for deoxy- and CO-hemoglobin (Hb), and for the HbCO photoproduct, obtained with varying delay between photolysis and probe laser pulses. At 10- to 20-mu-s delays, the photoproduct-HbCO difference spectrum shows numerous Tyr and Trp difference signals that are indicative of having reached the T state, on the basis of their likeness to the static deoxy Hb-HbCO difference spectrum. At earlier delays, the difference signals diminish in intensity and, at submicrosecond delays, are different in shape and frequency. Deconvolution analysis of the W3 band envelope and application of the correlation of W3 frequency with the dihedral angle, chi-2,1, to the X-ray coordinates facilitated assignment of these components to the three inequivalent Trp residues, beta-37, alpha-14, and beta-15. The Trp beta-37 assignment was confirmed via the UVRR spectrum of Hb Rothschild, in which Trp-beta-37 is replaced by arginine. The unique environmental sensitivity of the W3 frequency makes this band a particularly rich source of information in the time-resolved UVRR difference spectra, as the different W3 components exhibit quite different temporal evolutions. Changes in these components were evaluated in terms of environmental effects with the aid of excitation profiles (EP's) for the Trp model, 3-methylindole in various solvents. Prompt (30 ns) spectral changes were shown, on the basis of the W3 assignments, to be associated with displacements of the A and E helices on the distal side of the heme pocket in the immediate photoproduct. The 1-mu-s transient is characterized by an upshifted W17 band, which suggest an intermediate protein structure in which the Trp beta-37 R-state H-bond is broken. The Tyr-nu-8a and nu-8b (Y8a and Y8b) difference signals are shown to result from small upshifts, with a slight intensity loss in Y8a. These changes are detected in bands that result from six inequivalent but unresolvable Tyr residues. The crystal structures, however, indicate that the H-bonding environments of five of these residues differ insignificantly between the R and T states, and attention naturally focuses on the remaining Tyr-alpha-42 residue. This residue resides in the ''switch'' region of the alpha-1-beta-2 interface, where it forms an H-bond with the carboxylate side chain of Asp-beta-99 only in the T state. Elimination of this H-bond via mutations at Asp-beta-99 is known to reduce cooperativity by destabilizing the T state. It has long been assumed that Tyr-alpha-42 is the proton donor to a deprotonated Asp-beta-99 carboxylate in this H-bond. However, this should produce a downshift and intensification of Y8b on the basis of other proteins with donor Tyr-carboxylate H-bonds and of UVRR spectra of the Tyr model, p-cresol in solvents of varying donor and acceptor numbers. The observed Y8b upshift and loss of intensity in Y8a is instead consistent with Tyr-alpha-42 accepting a proton from a neutral Asp-beta-99 in this critical H-bond. An acceptor H-bond is readily accommodated by the crystallographic coordinates, which show the Asp-beta-99 side chain to be in a solvent-inaccessible hydrophobic environment. Whether this site can actually support protonation of carboxylate at neutral pH is discussed in the context of experimental and computational results.
