Ap. Campos et al., CYTOCHROME-C(6) FROM MONORAPHIDIUM-BRAUNII - A CYTOCHROME WITH AN UNUSUAL HEME AXIAL COORDINATION, European journal of biochemistry, 216(1), 1993, pp. 329-341
A soluble monoheme c-type cytochrome (cytochrome c6) has been isolated
from the green alga Monoraphidium braunii. It has a molecular mass of
9.3 kDa, an isoelectric point of 3.6 and a reduction potential of 358
mV at pH 7. The determined amino acid sequence allows its classificat
ion as a class-I c-type cytochrome. The ferric and ferrous cytochrome
forms and their pH equilibria have been studied using H-1-NMR, ultravi
olet/visible, EPR and Mossbauer spectroscopies. The pH equilibria are
complex, several pK(a) values and pH-dependent forms being observed. T
he amino acid sequence, the reduction-potential value and the visible
and NMR spectroscopies data in the pH range 4-9 indicate that the heme
iron has a methionine-histidine axial coordination. However, the EPR
and Mossbauer data obtained for the ferricytochrome show that in this
pH range two distinct forms are present: form I, g(z) = 3.27, g(y) = 2
.05 and g(x) = 1.05; form II, g(z) = 2.95, g(y) = 2.29 and g(x) = 1.43
. While form I has crystal-field parameters typical of a methionine-hi
stidine coordination, those associated with form II would suggest a hi
stidine-histidine axial ligation. This possibility was extensively ana
lyzed by spectroscopic methods and by chemical modification of a histi
dine residue. It was concluded that form II actually corresponds to an
unusual type of methionine-histidine axial coordination. Straightforw
ard examples of this type of coordination have recently been found in
other c-type hemeproteins [Teixeira, M., Campos, A. P, Aguiar, A. P.,
Costa, H. S., Santos, H., Turner, D. L. & Xavier, A. V. (1993) FEBS Le
tt. 317, 233 - 236], corroborating our proposal. Since both forms, wit
h very distinct crystal-field parameters, are shown to have the same r
eduction potential, it may be concluded that the axial and rhombic dis
tortions of the heme-iron ligand field cannot be directly correlated w
ith the heme-reduction potential. The pH-dependence studies have also
shown that the form I and form II are interconvertible, with pK(a) alm
ost-equal-to 5. To establish a possible physiological significance for
this process, in particular for the interaction of the cytochrome wit
h the membrane-bound electron-transfer complexes b6f and photosystem 1
, the effect of surfactants on the spectroscopic characteristics of cy
tochrome c6 has been studied.