UV resonance Raman spectra reveal a structural basis for diminished protonand CO2 binding to alpha,alpha-cross-linked hemoglobin

UV resonance Raman difference spectra between ligated and deoxyhemoglobin c ontain tryptophan and tyrosine signals which arise from quaternary H-bonds in the T state, which are broken in the R state. These H-bonds are unaffect ed by bis(3,5-dibromosalicyl) fumarate cross-linking at the Lys alpha 99 re sidues, which prevents dissociation of Hb tetramers to dimers. However, whe n the pH is lowered from 9.0, or when NaCl is added, intensity is diminishe d for the tyrosine Y8 and tryptophan W3 bands of cross-linked deoxyHb, but not of native deoxyHb. This effect is attributed to weakening of tertiary H -bonds involving Tyr alpha 140 and Trp alpha 14, when the T state salt brid ge between Val alpha 1 and Arg alpha 141 is formed via protonation of the t erminal amino group and anion binding. The Tyr alpha 140-Val alpha 93 H-bon d connects the Arg alpha 141-bearing H helix with the Lys alpha 99-bearing G helix. Weakening of the H-bond reflects a tension between the fumarate li nker and the salt-bridge. This tension inhibits protonation of the Val alph a 1 amino terminus, thus accounting for the diminution of both proton [Bohr effect] and CO2 binding in the T state as a result of cross-linking.