Modulation of ligand binding in engineered human hemoglobin distal pocket

Functional and structural studies on hemoglobin and myoglobin from differen t animals and engineered variants have enlightened the great importance of the physico-chemical properties of the side-chains at topological position B10 and E7. These residues proved to be crucial to the discrimination and s tabilisation of gaseous ligands. Ln view of the data obtained on the high o xygen affinity hemoglobin from Ascaris worms and a new mutant of sperm whal e myoglobin, we selected the two mutations Leu B10 --> Tyr and His E7 --> G in as potentially relevant to control ligand binding parameters in the alph a and beta-chains of human hemoglobin. Here, we present an investigation of three new mutants: Hb alpha YQ (alpha( 2)(YQ)beta(2)(A)), Hb beta YQ (alpha(2)(A)beta(2)(YQ)) and Hb alpha beta YQ (alpha(2)(YQ)beta(2)(YQ)). They are characterised by a very low reactivity for NO, O-2 and CO, and a reduced cooperativity. Their functional properti es are not inconsistent with the behaviour expected for a two-state alloste ric model. Proteins with these substitutions may be considered as candidate s for the synthesis of a possible "blood substitute", which should yield an O-2 adduct stable to autoxidation and slowly reacting with NO. The mutant Hb alpha beta YQ is particularly interesting because the rate of reaction o f NO with the oxy and deoxy derivatives is reduced. A structural interpreta tion of our data is presented based on the 3D structure of deoxy Hb alpha b eta YQ determined by crystallography at 1:8 Angstrom resolution. (C) 1999 A cademic Press.