Functional diversification within the heme-binding split-barrel family

Due to neofunctionalization, a single fold can be identified fi ed in multiple proteins that have distinct molecular functions. Depending on the time that has passed since gene duplication and the number of mutations, the sequence similarity between functionally divergent proteins can be relatively high, eroding the value of sequence similarity as the sole tool for accurately annotating the function of uncharacterized homologs. Here, we combine bioinformatic approaches with targeted experi- mentation to reveal a large multifunctional family of putative enzymatic and nonenzymatic proteins involved in heme metabolism. This family (homolog of HugZ (HOZ)) is embedded in the " FMN-binding split barrel" " superfamily and contains separate groups of proteins from prokaryotes, plants, and algae, which bind heme and either catalyze its degrada- tion or function as nonenzymatic heme sensors. In pro- karyotes these proteins are often involved in iron assimilation, whereas several plant and algal homologs are predicted to degrade heme in the plastid or regulate heme biosynthesis. In the plant Arabidopsis thaliana, , which contains two HOZ subfamilies that can degrade heme in vitro (HOZ1 and HOZ2), disruption of At HOZ1 (AT3G03890) or At HOZ2A (AT1G51560) causes developmental delays, pointing to important biological roles in the plastid. In the tree Populus trichocarpa, , a recent duplication event of a HOZ1 ancestor has resulted in localization of a paralog to the cytosol. Structural characterization of this cytosolic paralog and comparison to published homologous structures suggests conservation of heme-binding sites. This study unifies fi es our understanding of the sequence-structure-function relation- ships within this multilineage family of heme-binding pro- teins and presents new molecular players in plant and bacterial heme metabolism.