E light-harvesting like proteins (Lil proteins). In the genome of the cyanobacterium Synechocystis sp. PCC6803 (hereafter Synechocystis 6803), five lil genes have been identified, coding for proteins with high similarity to the plantFerrochelatase Refolding and KineticsFigure 1. Schematic representation of recombinant His-FeCh, FeCh, His-FeChD347 and FeChD347 of Synechocystis 6803. The C-terminal CAB domain is exclusive to plastidic ferrochelatases of photosynthetic organisms, it is connected via a linker region to the catalytical domain (amino acids 1-324), where chelating of divalent metal ions into protoporphyrin IX takes place. N-terminal His6-tags have been added with the amino acid sequence MGSSHHHHHHSSGLVPRGSH (for His-FeCh, cleavable by a thrombin protease) or MAHHHHHHVDDDDK (for His-FeChD347, cleavable by an enterokinase), respectively. doi:10.1371/journal.pone.0055569.glight-harvesting complexes [12]. Four genes encode the small CAB-like proteins (SCPs or high light induced proteins, HLIPs) referred to as ScpB-E, which have a molecular mass of around 6 kDa and have been shown to be involved in chlorophyll biosynthesis and the stabilization of chlorophyll-binding proteins [14,15,16,17]. The fifth gene, also referred to as ScpA, encodes the C-terminal part of the ferrochelatase enzyme. It has been suggested that the ancient ferrochelatase captured a membranespanning helix from a SCP/HLIP in order to fulfill functions for membrane Chebulagic acid anchoring or photoprotection of porphyrins [13]. Changes in the activity of the ferrochelatase have been shown to influence chlorophyll biosynthesis [18], and while inactivation of ScpA only has a subtle effect on enzyme activity [12], truncation of both ScpA and its linker segments impair enzyme activity [19]. Chl is the most abundant tetrapyrrole in plants and cyanobacteria, and the magnesium-chelatase and ferrochelatase enzymes compete for the same substrate, Protoporphyrin IX, for insertion of either magnesium for 
Chl biosynthesis or ferrous ion for heme biosynthesis, and in cyanobacteria also for phycobilin biosynthesis. However, the control step at the metal insertion branch point is poorly understood. While magnesium-chelatase comprises three subunits, CHLD, CHLI and CHLH [20] and requires ATP for activity, ferrochelatase is composed of a single subunit and requires no cofactors [2]. To guarantee a balanced flow of precursors in the pathway, the distribution of tetrapyrroles to the Fe- or Mg-branch, respectively, has to be tightly regulated. There may be up to 100 times more Chl in a cell than all other tetrapyrroles together [1]. It has therefore been suggested that 
Chl availability might positively regulate ferrochelatase activity [14,19]. The expression or activitiy of the chelatases have been studied by various research groups and factors that have been proposed as being important are e.g. I-BRD9 ATP-availability, redox state, enzyme localization, gene expression and substrate affinities [6,21,22,23,24]. In this paper we report a protocol for the functional refolding and purification from inclusion bodies, without truncation products or soluble aggregates, of recombinant Synechocystis 6803 ferrochelatase (FeCh). Enzyme kinetics were studied using Zn2+ and protoporphyrin IX as substrates for the monomeric form of FeCh that was either refolded from inclusion bodies, co-expressed with chaperones or lacking the CAB domain (FeChD347). We elucidated the effect of the C-terminal CAB-domain on theFigure 2.E light-harvesting like proteins (Lil proteins). In the genome of the cyanobacterium Synechocystis sp. PCC6803 (hereafter Synechocystis 6803), five lil genes have been identified, coding for proteins with high similarity to the plantFerrochelatase Refolding and KineticsFigure 1. Schematic representation of recombinant His-FeCh, FeCh, His-FeChD347 and FeChD347 of Synechocystis 6803. The C-terminal CAB domain is exclusive to plastidic ferrochelatases of photosynthetic organisms, it is connected via a linker region to the catalytical domain (amino acids 1-324), where chelating of divalent metal ions into protoporphyrin IX takes place. N-terminal His6-tags have been added with the amino acid sequence MGSSHHHHHHSSGLVPRGSH (for His-FeCh, cleavable by a thrombin protease) or MAHHHHHHVDDDDK (for His-FeChD347, cleavable by an enterokinase), respectively. doi:10.1371/journal.pone.0055569.glight-harvesting complexes [12]. Four genes encode the small CAB-like proteins (SCPs or high light induced proteins, HLIPs) referred to as ScpB-E, which have a molecular mass of around 6 kDa and have been shown to be involved in chlorophyll biosynthesis and the stabilization of chlorophyll-binding proteins [14,15,16,17]. The fifth gene, also referred to as ScpA, encodes the C-terminal part of the ferrochelatase enzyme. It has been suggested that the ancient ferrochelatase captured a membranespanning helix from a SCP/HLIP in order to fulfill functions for membrane anchoring or photoprotection of porphyrins [13]. Changes in the activity of the ferrochelatase have been shown to influence chlorophyll biosynthesis [18], and while inactivation of ScpA only has a subtle effect on enzyme activity [12], truncation of both ScpA and its linker segments impair enzyme activity [19]. Chl is the most abundant tetrapyrrole in plants and cyanobacteria, and the magnesium-chelatase and ferrochelatase enzymes compete for the same substrate, Protoporphyrin IX, for insertion of either magnesium for Chl biosynthesis or ferrous ion for heme biosynthesis, and in cyanobacteria also for phycobilin biosynthesis. However, the control step at the metal insertion branch point is poorly understood. While magnesium-chelatase comprises three subunits, CHLD, CHLI and CHLH [20] and requires ATP for activity, ferrochelatase is composed of a single subunit and requires no cofactors [2]. To guarantee a balanced flow of precursors in the pathway, the distribution of tetrapyrroles to the Fe- or Mg-branch, respectively, has to be tightly regulated. There may be up to 100 times more Chl in a cell than all other tetrapyrroles together [1]. It has therefore been suggested that Chl availability might positively regulate ferrochelatase activity [14,19]. The expression or activitiy of the chelatases have been studied by various research groups and factors that have been proposed as being important are e.g. ATP-availability, redox state, enzyme localization, gene expression and substrate affinities [6,21,22,23,24]. In this paper we report a protocol for the functional refolding and purification from inclusion bodies, without truncation products or soluble aggregates, of recombinant Synechocystis 6803 ferrochelatase (FeCh). Enzyme kinetics were studied using Zn2+ and protoporphyrin IX as substrates for the monomeric form of FeCh that was either refolded from inclusion bodies, co-expressed with chaperones or lacking the CAB domain (FeChD347). We elucidated the effect of the C-terminal CAB-domain on theFigure 2.
