The chlorophyll biosynthesis regulator GENOMES UNCOUPLED 4 (GUN4) is conserved in nearly all oxygenic photosynthetic organisms. Recently, GUN4 has been found to be able to bind the linear tetrapyrroles (bilins) and stimulate the magnesium chelatase activity in the unicellular green alga Chlamydomonas reinhardtii. Here, we characterize GUN4 proteins from Arabidopsis thaliana and the cyanobacterium Synechocystis sp. PCC 6803 for their ability to bind bilins, and present the crystal structures of Synechocystis GUN4 in biliverdin-bound, phycocyanobilin-bound, and phytochromobilin-bound forms at the resolutions of 1.05, 1.10, and 1.70 Å, respectively. These linear molecules adopt a cyclic-helical conformation, and bind more tightly than planar porphyrins to the tetrapyrrole-binding pocket of GUN4. Based on structural comparison, we propose a working model of GUN4 in regulation of tetrapyrrole biosynthetic pathway, and address the role of the bilin-bound GUN4 in retrograde signaling.

Arabidopsis thaliana heme oxygenase‐1 (AtHO‐1), a metabolic enzyme in the heme degradation pathway, serves as a prototype for study of the bilin‐related functions in plants. Past biological analyses revealed that AtHO‐1 requires ferredoxin‐NADP + reductase (FNR) and ferredoxin for its enzymatic activity. Here, we characterized the binding and degradation of heme by AtHO‐1, and found that ferredoxin is a dispensable component of the reducing system that provides electrons for heme oxidation. Furthermore, we reported the crystal structure of heme‐bound AtHO‐1, which demonstrates both conserved and previously undescribed features of plant heme oxygenases. Finally, the electron transfer pathway from FNR to AtHO‐1 is suggested based on the known structural information.