PROBING THE ALPHA(1)BETA(2) INTERFACE OF HUMAN HEMOGLOBIN BY MUTAGENESIS - ROLE OF THE FG-C CONTACT REGIONS

The allosteric transition of hemoglobin involves an extensive reorgani zation of the alpha(1) beta(2) interface, in which two contact regions have been identified. This paper concerns the effect of two mutations located in the ''switch'' (alpha C3 Thr --> Trp) and the ''flexible j oint'' (beta C3 Trp --> Thr). We have expressed and characterized one double and two single mutants: Hb alpha T38W/beta W37T, Hb beta W37T, and Hb alpha T38W, whose structure has been determined by crystallogra phy. We present data on: (i) the interface structure in the two contac t regions, (ii) oxygen and CO binding kinetics and cooperativity, (iii ) dissociation rates of deoxy tetramers and association rates of deoxy dimers, and (iv) the effect of NaI on deoxy tetramer dissociation rat e constant. All the mutants are tetrameric and T-state in the deoxygen ated derivative. Reassociation of deoxygenated dimers is not modified by interface mutations. DeoxyHb alpha T38W dimerizes 30% slower than H bA; Hb beta W37T and Hb alpha T38W/beta W37T dissociate much faster. W e propose a binding site for I- at the switch region. The single mutan ts bind O-2 cooperatively; the double one is almost non-cooperative, a feature confirmed by CO binding. The functional data, analyzed with t he two-state model, indicate that these mutations reduce the value of the allosteric constant L(0).