Isolation of Phytoflavin, A Flavoprotein with Chloroplast Ferredoxin Activity

During recent years considerable atteintion has been focused on the role of non-haem proteins containing iron in biological electron transfer systems. Several proteins of this type showing identical catalytic properties have been isolated from green cells (6). These proteins, which are localized in the chloroplasts, include the methaemoglobin reducing factor (5) from parsley, photosynthetic pyridine nucleotide reductase (17) from spinach, and the rotes Ferment (7) from Chlorella. All catalyze the photoreduction of NADP by chloroplasts. A protein possessing similar properties has been found in anaerobic bacteria. This protein has been named ferredoxin (14). Like the chloroplast protein, ferredoxin contains iron and sulfur as the only known constitutents other than protein. Ferredoxin functions as an electron carrier in nitrogen fixation (13), the synthesis (1) and cleavage (15) of pyruvate, formate oxidation (3), and in several hydrogenase coupled reactions including the reduction of hydroxylamine (24), NADP (22) and the oxidation of hypoxanthine (23). Ferredoxin also supports the photoreductioni of NADP by chloroplasts (21). Conversely, the related chloroplast protein can replace bacterial ferredoxin in the hydrogenase system of Clostridiuntii pasteuriantumi (21). For these reasons Tagawa anld Arnon (21) have proposed that the termi ferredoxini should also include the non-haem iron containing protein found in chloroplasts. This terminology will be adopted here, although it is to be noted that chloroplast and bacterial ferredoxins are not idenltical and differ in their absorption spectra, molecular weight and the number of labile iron and sulfur atoms per mole protein. The isolation of a new protein showing chloroplast ferredoxin activity is reported in this paper. This protein is not a ferredoxin. Since it contains FMN and mediates in the photoreduction of NADP by chloroplasts isolated from a variety of higher plants and algae, it has been named phytoflavin. The biological properties of phytoflavin will be described in a subsequent paper. A preliminary report of this work has appeared (20). Materials and Methods