Phenyl-deuterated tyrosine (Tyr-
d4) andindole-deuterated tryptophan (Trp-
d5) havebeenselectively incorporated into hemoglobin (Hb) by expressing the gene inauxotrophic strains of
Escherichiacoli. Ultraviolet resonance Ra
man (UVRR) spectra, using229-nm excitation, show that difference featurescharacteristic of the Hb quaternary R
![](/i<font color=)
mages/entities/rarr.gif"> T transition are notperturbed by the incorporation of the isotopes.All the UVRR bands between 800 and 1700 cm
-1 areassigned to either Tyr or Trp except for the 1511cm
-1 band, which had been thought to arise from the Trp 2× W18 overtone. This band does not shiftupon Trp or Tyr labeling but does shift 5 cm
-1 inD
2O, suggesting assignment to a histidine (His)residue.Its intensification in the T-state is consistent with Hisprotonation. The
![](/i<font color=)
mages/gifchars/alpha.gif" BORDER=0>- and
![](/i<font color=)
mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-subunits wereselectivelylabeled, by reconstitution of labeled subunits with unlabeled subunits,to
make isotope hybrids. SelectiveTyr labeling identified the
![](/i<font color=)
mages/gifchars/alpha.gif" BORDER=0> subunits as the locus of the Y8aupshift observed in Hb, supporting theprevious inference that this shift is associated with the T-stateH-bond involving the interfacial Tyr
![](/i<font color=)
mages/gifchars/alpha.gif" BORDER=0>42[Rodgers, Su, Subra
maniam, & Spiro (1992)
J.
Am.
Chem.
Soc.
114, 3697]. SelectiveTrp labeling showedthe Trp
![](/i<font color=)
mages/gifchars/alpha.gif" BORDER=0>14 contributions to the T - R difference spectrum to benegligible and confirmed Trp
![](/i<font color=)
mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">37 asthe locus of the W3 difference signal, and probably of the re
mainingTrp signals as well. The observeddownshift of W17 and upshift of Wd5 in the T-state are consistent witha stronger T-state H-bond betweenTrp
![](/i<font color=)
mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">37 and Asp
![](/i<font color=)
mages/gifchars/alpha.gif" BORDER=0>94; the resulting excitation profile red shiftaccounts for the dominance of the Trp
![](/i<font color=)
mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">37contribution to the T - R difference UVRR spectrum.