Curcumin, bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, is a natural yellow-orange dye derived from the rhizome of Curcuma longa, an East Indian plant. In order to understand the photobiology of curcumin better we have studied the spectral and photochemical properties of both curcumin and 4-(4-hydroxy-3-methoxy-phenyl)-3-buten-2-one (hC, half curcumin) in different solvents. In toluene, the absorption spectrum of curcumin contains some structure, which disappears in more polar solvents, e.g. ethanol, acetonitrile. Curcumin fluorescence is a broad band in acetonitrile (lambda max = 524 nm), ethanol (lambda max = 549 nm) or micellar solution (lambda max = 557 nm) but has some structure in toluene (lambda max = 460, 488 nm). The fluorescence quantum yield of curcumin is low in sodium dodecyl sulfate (SDS) solution (phi = 0.011) but higher in acetonitrile (phi = 0.104). Curcumin produced singlet oxygen upon irradiation (lambda > 400 nm) in toluene or acetonitrile (phi = 0.11 for 50 microM curcumin); in acetonitrile curcumin also quenched 1O2 (kq = 7 x 10(6) M-1 s-1). Singlet oxygen production was about 10 times lower in alcohols and was hardly detectable when curcumin was solubilized in a D2O micellar solution of Triton X-100. In SDS micelles containing curcumin no singlet oxygen phosphorescence could be observed. Curcumin photogenerates superoxide in toluene and ethanol, which was detected using the electron paramagnetic resonance/spin-trapping technique with 5,5-dimethyl-pyrroline-N-oxide as a trapping agent. Unidentified carbon-centered radicals were also detected.(ABSTRACT TRUNCATED AT 250 WORDS)

The Cercospora nicotianae SOR1 (singlet oxygen resistance) gene was identified previously as a gene involved in resistance of this fungus to singlet-oxygengenerating phototoxins. Although homologues to SOR1 occur in organisms in four kingdoms and encode one of the most highly conserved proteins yet identified, the precise function of this protein has, until now, remained unknown. We show that SOR1 is essential in pyridoxine (vitamin B6) synthesis in C. nicotianae and Aspergillus flavus, although it shows no homology to previously identified pyridoxine synthesis genes identified in Escherichia coli. Sequence database analysis demonstrated that organisms encode either SOR1 or E. coli pyridoxine biosynthesis genes, but not both, suggesting that there are two divergent pathways for de novo pyridoxine biosynthesis in nature. Pathway divergence appears to have occurred during the evolution of the eubacteria. We also present data showing that pyridoxine quenches singlet oxygen at a rate comparable to that of vitamins C and E, two of the most highly efficient biological antioxidants, suggesting a previously unknown role for pyridoxine in active oxygen resistance.The filamentous, phytopathogenic fungus Cercospora nicotianae exhibits a uniquely effective, broad-spectrum resistance to potent photosensitizers of diverse chemical structure and solubility (1, 2). C. nicotianae is resistant to cercosporin, a light-activated, singlet oxygen ( 1 O 2 )-generating toxin it produces in culture and during plant parasitism, and also to other potent photosensitizers including porphyrins and xanthine and thiazine dyes. Photosensitizers are highly toxic compounds that produce their deleterious effects only after activation by light. Absorbed light energy converts the photosensitizer to an excited (triplet) state molecule that may transfer an electron to oxygen to generate superoxide and͞or transfer energy directly to oxygen, yielding 1 O 2 (3). Exposure of cells to photosensitizers plus light leads to the destruction of critical cellular components including proteins, membranes, and DNA and often results in cell death. Studies on the mechanisms by which organisms protect themselves against reactive oxygen species have focused primarily on reduced and radical forms of oxygen, including hydrogen peroxide (H 2 O 2 ), superoxide (O 2 . ), and the hydroxyl radical (OH⅐). These active oxygen species are byproducts of normal cellular metabolism, and cells contain numerous and conserved defenses against them. By contrast, the highly reactive, but nonradical 1 O 2 is produced primarily via light activation of photosensitizing compounds. Most organisms do not tolerate 1 O 2 , and few biological defenses have been identified (2). The broad-spectrum resistance expressed by Cercospora species against cercosporin and other photosensitizers of diverse structure make these organisms an excellent model for understanding the cellular basis of 1 O 2 resistance. To study specific genes and proteins involved in photosensitizer and 1 O 2 resistance...

The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is frequently used to identify free radicals that are generated photochemically using dyes as photosensitizers. When oxygen is present in such systems, singlet oxygen (1O2) may be produced and can react with DMPO. We have studied the reaction of DMPO with 1O2 in aqueous solutions over a wide range of pH, using micellar Rose Bengal (pH 2−13) and anthrapyrazole (pH < 2) as photosensitizers. We found that DMPO quenches 1O2 phosphorescence (k q = 1.2 × 106 M-1 s-1), thereby initiating oxygen consumption that is slow at pH 10 but increases about 10-fold at pH < 6. This oxygen consumption is a composite process that includes efficient oxidation of both DMPO and its degradation products. The oxidation products include both products in which the DMPO pyrroline ring remains intact (DMPO/•OH and 5,5-dimethyl-2-oxo-pyrroline-1-oxyl (DMPOX) radicals) and those in which it becomes opened (nitro and nitroso products). The nitroso product itself strongly quenched 1O2 phosphorescence, while (photo)decomposition of the nitroso group, presumably to nitric oxide (NO•), produced nitrite as a minor product. We propose that 1O2 adds to the >CN(O) bond in DMPO, producing a biradical, >C(OO•)N•(O). This biradical may follow one of two pathways:  (i) It may be protonated and rearrange to a strongly oxidizing nitronium-like moiety, which could be reduced to the DMPO hydroperoxide radical DMPO/•O2H while oxidizing another DMPO moiety to ultimately form DMPOX. The DMPO/•O2H could undergo further redox decomposition, e.g. via the known Fenton-like reaction, to produce both free •OH radical and the DMPO/•OH radical. (ii) The biradical >C(OO•)N•(O) may cyclize to a 1,2,3-trioxide (ozonide), which could open the pyrroline ring to form 4-methyl-4-nitropentan-1-al and 4-methyl-4-nitrosopentanoic acid. Because the oxidation of DMPO by 1O2 leads to both rapid O2 depletion and the formation of transients and products that might interfere with trapping and identification of free radicals, DMPO should be used with caution in systems where 1O2 is produced.

Vitamin B6 (pyridoxine, 1) and its derivatives: pyridoxal (2), pyridoxal 5‐phosphate (3) and pyridoxamine (4) are important natural compounds involved in numerous biological functions. Pyridoxine appears to play a role in the resistance of the filamentous fungus Cercospora nicotianae to its own abundantly produced strong photosensitizer of singlet molecular oxygen (1O2), cercosporin. We measured the rate constants (kq) for the quenching of 1O2 phosphorescence by 1–4 in D2O. The respective total (physical and chemical quenching) kq values are: 5.5 × 107M−1 s−1 for 1; 7.5 × 107M−1 s−1 for 2, 6.2 ×107M−1 s−1 for 3 and 7.5 × 107M−1 s−1 for 4, all measured at pD 6.2. The quenching efficacy increased up to five times in alkaline solutions and decreased ∼10 times in ethanol. Significant contribution to total quenching by chemical reaction(s) is suggested by the degradation of all the vitamin derivatives by 1O2, which was observed as declining absorption of the pyridoxine moiety upon aerobic irradiation of RB used to photosensitize 1O2. This photodegradation was completely stopped by azide, a known physical quencher of 1O2. The pyridoxine moiety can also function as a redox quencher for excited cercosporin by forming the cercosporin radical anion, as observed by electron paramagnetic resonance. All B6 vitamers fluoresce upon UV excitation. Compounds 1 and 4 emit fluorescence at 400 nm, compound 2 at 450 nm and compound 3 at 550 nm. The fluorescence intensity of 3 increased ∼10 times in organic solvents such as ethanol and 1,2‐propanediol compared to aqueous solutions, suggesting that fluorescence may be used to image the distribution of 1–4 in Cercospora to understand better the interactions of pyridoxine and 1O2 in the living fungus.