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.