PURIFICATION AND SPECTROSCOPIC CHARACTERIZATION OF A RECOMBINANT CHLOROPLASTIC HEMOGLOBIN FROM THE GREEN UNICELLULAR ALGA CHLAMYDOMONAS-EUGAMETOS

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.