M. Couture et M. Guertin, PURIFICATION AND SPECTROSCOPIC CHARACTERIZATION OF A RECOMBINANT CHLOROPLASTIC HEMOGLOBIN FROM THE GREEN UNICELLULAR ALGA CHLAMYDOMONAS-EUGAMETOS, European journal of biochemistry, 242(3), 1996, pp. 779-787
Hemoglobins (Hb), which have the important task of delivering molecula
r oxygen by facilitating its reversible binding to the heme, are now t
hought to have evolved in all groups of organisms including prokaryote
s, fungi, plants and animals. Our recent finding of a light-inducible
chloroplastic Hb in the green unicellular alga Chlamydomonas eugametos
has further extend this idea, while raising questions about the funct
ion that an Hb could play in a high oxygen environment such as in the
chloroplast. In order to understand the role played by this new Hb, we
have undertaken its biochemical characterization. To facilitate the c
haracterization of Chlamydomonas Hb, which represents less than 0.01%
of the soluble protein in the green alga, the protein has been express
ed in Escherichia coli and purified to apparent homogeneity. The purif
ied recombinant protein possesses a non-covalently bound iron-protopor
phyrin IX heme. The oxy form of the recombinant Hb, purified directly
from bacterial cells, is very stable, with a measured half-life of 7 d
ays at pH 8 and has an ultraviolet/visible spectrum similar to those o
f the related cytoplasmic Hbs of the ciliated protozoa Paramecium and
Tetrahymena and of the cyanobacterium Nostoc commune. In contrast to w
hat has been reported for oxymyoglobins and oxyhemoglobins, the dioxyg
en molecule bound to the LI637 Hb can be reduced by the electron-trans
fer mediator phenazine methosulfate in the presence of NADPH, indicati
ng that the heme pocket of Chlamydomonas Hb may be more accessible to
small molecules. With regard to this, we found that when the small red
ucing agent sodium dithionite is used to reduce the met form, it must
be removed anaerobically from the Hb prior to oxygenation of the prote
in to stably produce the oxy form. Otherwise, the oxy form is obtained
readily from the met form under an oxygenic atmosphere when ferredoxi
n and ferredoxin NADP(+) reductase are used to enzymically reduce the
Hb. Finally, the spectra of the deoxy and met forms were unusual, the
heme being partly low-spin at physiological pH. These results confirm
the existence of a reversible oxygen-binding protein in the chloroplas
t of C. eugametos. The unusual spectral and biochemical properties of
the protein may reflect a specialized function for this Hb.