@misc{oai:ir.soken.ac.jp:00000261,
author = {目, 喜直 and サカン, ヨシナオ and SAKAN, Yoshinao},
month = {2016-02-17, 2016-02-17},
note = {Myoglobin(Mb) that is monomer and Hemoglobin(Hb) that is
tetramer are one of the most basic hemoproteins. Hb has a Physiological
function as an oxygen carrier in blood, and Mb can store oxygen in mus-
cle. Both of Mb and a subunit of Hb consist of one heme (iron protopor-
phtrin IX) and a single polypeptide chain.Mb and Hb bind carbonmonox-
ide(CO) at the oxygen binding site. Carbonmonoxy myoglobin (MbCO)
and carbonmonoxy hemoglobin (HbCO) are photodissociable upon laser
illumination,reversibly.
According to the x-ray data of crystalline Mb,there is no pathway
for the ligand entry from the solvent to the heme pocket. However,in
some of derivative Mb another structure was also detected, in which distal
histidine swings out toward the solvent and a pathway for the ligand entry
is open. This structure is called "open"form, and the other structure that
has no pathway is called "closed" form. When the ligand enters into the
heme pocket , it is expected that the protein around the heme undergoes
the conformational change. On the other hand, IR and Raman spectros-
copy showed that in acidic MbCO, the C-O(vc-o) and Fe-CO(vFe-co)
stretchinig vibrational modes are located at different frequencies from
these of neutral MbCO. In the present Raman experiment two vFe-co
bands were identified at around 490 cm-1 and 510 cm-1 for MbCO at pH
4.5, while only one peak was observed at 510 cm-1 for MbCO at neutral
pH. The value pH 4.5 happens to be equal to the pKa value of the distal
histidine and thus, half of the distal histidine is protonated at pH 4.5.
Therefore, it was inferred that the protonated distal histidine repelled
lysine-45 in the vicinity of the heme distal side, and the protein conforma-
tion changed to the "open" form. Thus, the higher and lower frequency of
vFe-co correspond to the "closed" and "open" form, respectively. The vFe-
cofrequency is sensitive to the Fe-C-O angle. According to the XANES
of MbCO solution at neutral pH, the Fe-C-O bent angle is 150. From a
simple normal-coordinate calculation for isolated three-body oscillators,
the vFe-co frequencies for the bent angle of 160 and 180 were estimated
at 500 cm-1 and 490 cm-1, respectively. It is assumed that the Fe-C-O
angle of the "open" form is linear and that of "closed" form is bent.
If the photodissociated ligand recombines to the "open" form and
relaxes to the "closed" form gradually at neutral pH, the vFe-co should
appear around 490 cm-1 first and shift to 505 cm-1. In this study, the
nanosecond time-resolved resonance Ranman(TR3) spectra were ob-
served in order to catch the transient species of MbCO on the recombina-
tion reaction. TR3 spectroscopy is a powerful technique to analyze the
dynamical conformation in short time scale, and information obtained by it
is very basic and important in studies of the relationship between physio-
logical functions and protein dynamics.
MbCO at acidic pH(4.5), containing equal amounts of the "closed"
and the "open" forms, were measured by the TR3 system. If the assump-
tion above is true, the recombination rate of the "open" form should be
faster than that of the "closed" form. Indeed, the temporal behaviors of the
vFe-co bands of the "open" and "closed" forms were different. The vFe-co
band of the "open" form recovered faster than the "closed" form. Howev-
er, at neutral pH, there were no transient bands around 490 cm-1 in all
time range observed (-20ns-1ms). It suggests either that the transient
"open" form is absent or that the structural is change too fast to be detect
ed(ChapterII).
In the acide pH,the recombination kinetics may be affected by pH
effect. It is desirable to measure both of "open" and "closed" forms under
identical conditions including pH and temperatures. The human abnormal
hemoglobins, "Boston" and "Saskatoon" are best models for this purpose,
since abnormal hemoglobins contain two types subunits in one molecule,
that is normal chains and abnormal chains. In the abnormal chain the
distal histidine is replaced by tyrosine. In "Boston", the α-chain is abnor-
mal, and in "Saskatoon" the β-chain is abnormal. Their vFe-co bands
arise around 490 and 505 cm-1, corresponding to the "open" and "closed"
forms, respectively (ChapterIII).
Some human MbCO mutants whose distal His was replaced by
various amino acids residues through site-directed mutagenesis were
also examined at neutral pH in order to make clarity the role of the distal
histidine on the rebinding reaction. Some of them have the vFe-co band
around 490 cm-1 and the others have it around 510 cm-1 (ChapterIV).
The results from the experiments on these Hbs and Mbs also showed that
MbCO with the lower vFe-co band recovered faster than that with higher
band. Transient bands observed the time range between 100-1000ns
were significantly broad. The transient "open" form was not detected in all
time range for the species with the "closed" equilibrium structure. These
results suggested that the ligand enterd to the heme pocket through a
pathway which was created by a conformational changes in the distal side,
but the so-called "open" form was never produced during the recombina-
tion reaction. The Fe-C-O angle in a transient form seems to be slightly
perturbed around the equilibrium angle and it has appreciable distribu-
tions., application/pdf, 総研大甲第36号},
title = {時間分解共鳴ラマン分光法による光解離後の一酸化炭素結合型ミオグロビンとヘモグロビンのタンパク質動的構造の研究},
year = {}
}