Changes of near-UV CD spectrum of human hemoglobin upon oxygen binding: A study of mutants at (alpha) 42, (alpha)140, (beta)145 tyrosine or (beta)37 tryptophan

The deoxy-form of human adult hemoglobin (Hb A) exhibits a distinct negative CD band at 287 nm that disappears in the oxy-form. It has been suggested that the negative CD band is due to the environmental alteration of Tyr-(alpha)42 or Trp-(beta)37 at the(alpha) 1(beta)2 contact upon deoxygenation. To evaluate the contributions of the aromatic residues at the(alpha) 1(beta)2 contact and the penultimate tyrosine residues of the(alpha) and (beta) subunits ((alpha)140 and (beta) 145) to the negative CD band, three recombinant (r) Hbs (rHb Ser-(alpha) 42, rHb His-(beta)37, and rHb Thr-(beta)145) were produced in Escherichia coli, and we compared the near-uv CD spectra of these three rHbs and Hb Rouen (Tyr-(alpha)140(right arrow) His) with the spectra of Hb A under the condition in which all mutant Hbs were able to undergo the T(right arrow) R transition (Hillʹs n > 2.0). The contributions of Tyr-(alpha) 42, Trp-(beta)37, Tyr-(alpha)140, and Tyr-(beta)145 to the negative CD band were estimated from changes in the ellipticity of the negative CD band at 287 nm to be 4, 18, 32, and 27%, respectively. These results indicate that environmental alteration of the penultimate tyrosine residues caused by the formation of salt bridges upon the R(right arrow) T transition is primarily responsible for the negative CD band.