J. Biol. Chem., Vol. 261, Issue 24, 10976-10989, Aug, 1986
A study of roles of evolutionarily invariant proline 30 and glycine 34 of cytochrome c
E Poerio, GR Parr and H Taniuchi
The previous studies (Juillerat, M. A., and Taniuchi, H. (1986) J. Biol.
Chem. 261, 2697-2711), using a three-fragment complex (1-25)H X (28-38) X
(39-104) of horse cytochrome c, have shown that invariant leucine 32 and
partially invariant leucine 35, both buried in the interior, exhibit a
striking difference in perturbation of binding fragment (28-38) by
substitution with isoleucine. Then the idea has been proposed that the
energy states of leucine 32, the Met-80-S-heme- Fe bond and other distant
residues such as tryptophan 59 would be coupled to generate extra force
while leucine 35 would be less important for such coupling if it were
involved. In the present studies we synthesized three (28-38) analogs
substituting invariant proline 30 with glycine or invariant glycine 34 with
alanine or serine. Thermodynamic and kinetic studies and UV CD and
biological activity measurements were carried out on binding of the analogs
to complex (1- 25)H X (39-104). The results with the ferric form show that
perturbations of delta G, delta H, and delta S associated with formation of
the intermediate complex and with the ensuing process by the Gly34----Ala
or Ser substitution result in weakening the Met-80-S- heme-Fe bond formed
in the latter process; in contrast, perturbation by the Pro30----Gly
substitution is small. However, the biological activity is more perturbed
by the Pro30----Gly substitution than by the Gly34----Ala or Ser; and in
the Gly34----Ala or Ser substitution the complex appears to be more readily
activated for both formation and disruption of the Met-80-S-heme-Fe bond at
20 degrees C and below than without substitution. In all cases reduction of
the heme strengthens the binding of fragment (28-38). However, striking are
the increases in perturbation (less negative) of both delta H and delta S
for binding of fragment (28-38) to form the ground state on reduction of
the heme in the Pro30----Gly, Gly34----Ala or Ser (the present studies),
and Leu32-- --norvaline (the previous studies) substitutions. It is known
that fluctuation of the atomic positions of most residues of tuna
ferrocytochrome c including Pro30, Leu32, and Gly34 increases on oxidation
of the heme and that these three residues are among those showing the least
fluctuating atomic positions (Takano, T., and Dickerson, R.E. (1982) in
Electron Transport and Oxygen Utilization (Ho, C., ed) pp. 17-26,
Elsevier/North-Holland Biomedical Press, New York).(ABSTRACT TRUNCATED AT
400 WORDS)
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