Bilin-dependent regulation of chlorophyll biosynthesis by GUN4

Biosyntheses of chlorophyll and heme in oxygenic phototrophs share a common trunk pathway that diverges with insertion of magnesium or iron into the last common intermediate, protoporphyrin IX. Since both tetrapyrroles are pro-oxidants, it is essential that their metabolism is tightly regulated. Here, we establish that heme-derived linear tetrapyrroles (bilins) function to stimulate the enzymatic activity of magnesium chelatase (MgCh) via their interaction with GENOMES UNCOUPLED 4 (GUN4) in the model green alga Chlamydomonas reinhardtii. A key tetrapyrrole-binding component of MgCh found in all oxygenic photosynthetic species, CrGUN4, also stabilizes the bilin-dependent accumulation of protoporphyrin IX-binding CrCHLH1 subunit of MgCh in light-grown C. reinhardtii cells by preventing its photooxidative inactivation. Exogenous application of biliverdin IXα reverses the loss of CrCHLH1 in the bilin-deficient heme oxygenase (hmox1) mutant, but not in the gun4 mutant. We propose that these dual regulatory roles of GUN4:bilin complexes are responsible for the retention of bilin biosynthesis in all photosynthetic eukaryotes, which sustains chlorophyll biosynthesis in an illuminated oxic environment.

Isolation, characterization, and antioxidative activity of a dioxobilin-type phylloxanthobilin from savoy cabbage

The degradation of the green pigment chlorophyll in plants is known to yield phyllobilins as highly abundant linear tetrapyrroles. Recently, a split path of the degradation pathway has been discovered, leading to so-called dioxobilin-type (or type-II) phyllobilins. The first characterized type-II phyllobilin was colorless featuring four deconjugated pyrrole units. Similar to the type-I branch, for which yellow oxidation products of the colorless phyllobilins – the type-I phylloxanthobilins – are known, a type-II phylloxanthobilin has recently been characterized from senescent leaves of grapevine. Type-I phylloxanthobilins appear to be actively produced in the plant, are known to possess interesting chemical properties, and were shown to act as potent antioxidants that can protect cells from oxidative stress. Here we report the isolation and structural characterization of a type-II phylloxanthobilin from de-greened leaves of savoy cabbage, which turned out to be structurally closely related to bilirubin. Bilirubin is known to possess high antioxidative activity; in addition, savoy cabbage is considered to promote health benefits due to its high content in antioxidants. We therefore investigated the in vitro antioxidative potential of the newly identified type-II phylloxanthobilin using two different approaches, both of which revealed an even higher antioxidative activity for the type-II phylloxanthobilin from savoy cabbage compared to bilirubin.

Coupled oxidation of iron tetraarylporphyrins as a synthetic tool for linear tetrapyrroles

Coupled oxidation of iron tetraphenylporphyrins was studied as a tool for preparation of various linear tetrapyrroles. We obtained two linear tetrapyrrole products, biladienone with the meso carbon retained and bilindione with one of the meso carbons eliminated. Biladienone was isolated as a 15-hydroxy derivative. Acid catalyzed elimination of water from biladienone yielded bilatrienone. Bilindione was converted to the zinc complex of 5-oxaporphyrin by reacting it with acetic anhydride and zinc acetate. The zinc complex of 5-oxaporphyrin was a labile electrophile, and readily reacted with a weak nucleophile such as methanol or ethanol. The nucleophilic ring cleavage was used to prepare a variety of bilinone derivatives with substituent at the terminal pyrrole ring. The zinc complex of 5-oxaporphyrin fluoresced red light with a quantum yield of 7%. Free base bilinone showed solvatochromism by forming proton-transferred hydrogen bonding with aprotic amide, which bears similarity to the red to far-red photochromism of phytochromes. The zinc complex of biladienone bearing long alkyl chains was used as an active layer of molecular switch, where flexible framework of linear tetrapyrrole and self-assembling properties lead to 103 fold electric current reduction by application of an external electric field. The zinc complex of biladienone showed allosteric binding to amines, where allosteric behavior was caused by its dimerization.