JBC, Vol. 250, Issue 17, 6679-6685, Sep, 1975
Ethylisocyanide equilibria of hemoglobins M Iwate, M Boston, M Hyde Park, M Saskatoon, and M Milwaukee-I in half-ferric and fully reduced states
K. Nishikura, Y. Sugita, M. Nagai and Y. Yoneyama
The ethylisocyanide equilibria of all the five known hemoglobins M, namely
Hb M Iwate (alpha287 Tyrbeta2), Hb M Boston (alpha258 Tyrbeta2), Hb M Hyde
Park (alpha2beta292 Tyr), Hb M Saskatoon (alpha2beta263 tyr), and Hb M
Milwaukee-I (alpha2beta267 Glu), were studied both in the half-ferric and
fully reduced heme states. In the half-ferric state, no heme-heme
interaction was observed for Hb M Iwate, Hb M Boston, and Hb M Hyde Park,
but Hb M Saskatoon and Hb M Milwaukee-I show small but definite heme-heme
interaction with Hill's n of 1.3. The beta chain mutants, Hb M Hyde Park
and Hb M Saskatoon, have almost normal affinity for ethylisocyanide and a
normal Bohr effect, whereas the alpha chain mutants, Hb M Iwate and Hb M
Boston, have abnormally low affinity and almost no Bohr effect. Hb M
Milwaukee-I showed a large Bohr effect and low affinity. These results are
consistent qualitatively with those on oxygen equilibria reported
previously. In the fully reduced state, in which all four hemes were in the
ferrous state and capable of binding ethylisocyanide distinct differences
were found in the extent of heme-heme interaction. Namely, the n values for
proximal histidine mutants, Hb M Iwate and Hb M Hyde Park, were 1.1 and
1.0, respectively, whereas the distal histidine mutants, Hb M Boston and Hb
M Saskatoon, showed high n values of 2.4 and 1.6, respectively. Hb M
Milwaukee-I also exhibited a high n value of 2.0 The ethylisocyanide
affinity of the four histidine mutants was high compared with that of Hb A,
while that for Hb M Milwaukee-I was almost normal. All five Hbs M had
approximately normal magnitudes of Bohr effect. In the half-ferric state,
the proximal and distal histidine mutants of the same chain showed similar
affinity for ethylisocyanide and Bohr effect, rather different from those
of the mutants of the opposite chain. These differences seem to be derived
from the difference of abnormal bonding of ferric iron to tyrosine or
glutamic acid. On the other hand, the reduction of iron, which abolished
the abnormal bonding and made all of the chains capable of binding ligand,
extinguished the differences of alpha and beta chains, and the effect of
amino acid side chains close to iron on ligand binding properties became
clear. Proximal histidine, which is considered to trigger the transition
between the T and R states, seems to be essential to the heme-heme
interaction.
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