Singlet Oxygen-mediated Photobleaching of the Prosthetic Group in Hemoglobins and C-Phycocyanin

Abstract

Proteins bearing colored prosthetic groups, such as the heme group in hemoglobin or the bilin group in c-phycocyanin, quench singlet oxygen by interactions at the apoprotein and the prosthetic group levels. In both proteins, chemical modification of the chromophore constitutes only a minor reaction pathway. While total deactivation of singlet oxygen takes place with rate constants of 4.0 x 10(9) and 4.2 x 10(8) M-1 s-1 for hemoglobin and phycocyanin, respectively, the bleaching of the chromophore takes place with rate constants of 3.2 x 10(6) and approximately 1 x 10(7) M-1 s-1. Irradiation of phycocyanin with red light bleaches the chromophore with low yields (approximately 0.8 x 10(-4)). Part of this bleaching is mediated by singlet oxygen produced by the irradiation of the bilin group. The low relevance of the singlet oxygen pathway is compatible with a low quantum yield (approximately 10(-3)) of free singlet oxygen production after irradiation of the protein.

CpcM Posttranslationally Methylates Asparagine-71/72 of Phycobiliprotein Beta Subunits in Synechococcus sp. Strain PCC 7002 and Synechocystis sp. Strain PCC 6803

“…The ␤-81/82 PCB chromophores are more exposed in PC and AP of the cpcM mutant strains because of the absence of the ␥-N-methyl group on Asn72 (1). It has been reported that PBP can sensitize the formation of reactive oxygen species (25,26,63) and that PBP are sensitive to bleaching by reactive oxygen species (21,60,63). In future studies we will determine whether PBP lacking methylation at Asn71/72 produce more reactive oxygen species, whether the PBP themselves are more sensitive to reactive oxygen species, or both.…”

Phycobilins and Phycobiliproteins Used in Food Industry and Medicine

“…Various in vitro and in vivo studies have confirmed that phycocyanin and phycocyanobilin are efficient scavengers of ROS [140]. For instance, phycocyanin scavenges hydroxyl [140][141][142][143][144], alkoxyl radicals [141], and peroxyl radicals [117,[142][143][144][145][146][147], singlet oxygen [144,148], hydrogen peroxide [144,147], superoxide anions [144], hypochlorous acid [143,144,149] and reactive nitrogen species (RNS) such as peroxynitrite [144,150] and nitric oxide [151]. Phycobiliproteins (especially Se-containing phycocyanin) are also able to scavenge non-natural radicals (like e.g., 2,2-azinobis(3-ethylbenzothiazolin-6-sulphonic acid) [152,153].…”

“…In another in vitro study, phycocyanobilin was shown to be mostly responsible for the antioxidant activity of phycocyanin isolated from fresh or spray-dried Spirulina platensis against oxidation of methyl linoleate in a hydrophobic system or in phosphatidylcholine liposomes [156]. Obviously, the phycocyanobilin pigment(s) present in the phycobiliprotein complexes are involved in the process of ROS scavenging [142,146,148,152,154,157] as demonstrated clearly by their radical assisted bleaching during this process [142,148,154]. For instance, singlet oxygen and peroxyl radicals are stabilized by oxidizing the double bond of the tetrapyrrole [142,144,149].…”

“…However, heterologously expressed allophycocyanin apoprotein and even its subunits were shown to inhibit hydroxyl radicals [135], and both phycocyanobilin and the recombinant apoprotein were shown to take part in the quenching of singlet oxygen [148]. Similarly, both the native phycocyanin and the recombinant phycocyanin apoprotein (β subunit) were able to scavenge peroxyl radicals and hydrogen peroxide and to protect erythrocytes from oxidative damage, although the latter was less effective [147].…”

“…Methionine and cysteine [160,161] (and other) amino acid residues of the protein may contribute to its antioxidant effect (for instance hypochloric acid scavenging) [149] as demonstrated also by studies on isolated phycocyanin peptides [161]. Red light illumination of phycocyanin caused bleaching of phycocyanobilin due to the singlet oxygen production induced by the irradiation of the bilin group [148]. On the other hand, blue light increased the in vitro scavenging effect of phycocyanin due to changes induced in the conformation of the protein (altering the localization of cysteine residues potentially involved in ROS scavenging) [160,161].…”