Cystis 6803 was expressed in E. coli as inclusion bodies, unless coexpressed with the chaperones GroEL/GroES, while a construct depleted of this CAB motif, His-FeChD347, could be expressed fully soluble in E. coli. The use of standard purification protocols for purification of His-FeCh resulted either in truncation products or soluble aggregates, despite the usage of specialized E. coli strains (e.g. Rosetta2 (DE3)). The site of truncation was located at the Cterminal part of His-FeCh as revealed by immunoblotting and peptide mass fingerprinting (data not shown). We developed a protocol for the purification and refolding of recombinant ferrochelatase in order to circumvent these problems, and here we are able to show that 
recombinant full-length FeCh of Synechocystis 6803 is active as a monomer. In its monomeric state FeCh activity was dependent on the presence of potassium ions to stabilize the native protein structure through an apparent weak kosmotropic effect [30]. Recombinant FeCh activity was found to be insensitive to pH changes in the range of pH 6?, indicating additional regulation factors in vivo for this enzyme in photosynthetic organisms. Type I ferrochelatases are known to be regulated by the redox state of the cell [5], their activity was found to increase in response to environmental stresses, while type II activity is repressed under these conditions [5]. The temperature optimum of FeCh activity was at 30uC, coinciding with the typical growth temperature of Synechocystis 6803. At 37uC there was still appreciable activity of the enzyme, which then declined rapidly at higher temperatures (Fig. 4). The choice of detergent appears to be important for FeCh activity. Attachment to the photosynthetic membranes is required for type II ferrochelatases in vivo in order to pursue both uptake ITI007 ofProto9 and release of heme [27]. b-DM forms oblate micelles mimicking a biological membrane, while CHAPS micelles have a prolate shape [34,35]. Therefore b-DM seems to be better suited for optimal activity of FeCh compared to CHAPS. The lag phase, resulting in a 
sigmoidal progress curve that was observed when measuring FeCh activity in the presence of CHAPS, could be abolished by pre-incubating the enzyme with metal ions before the start of the assay. The increased activity after the lag phase therefore was not due to a decreasing zinc pool. Enzyme kinetic plots revealed cooperativity of FeCh and FeChD347 regarding Zn2+, the substrate metal therefore might bind to peripheral sites of the enzymes [36,37]. This cooperativity was even more pronounced studying the His-tagged enzymes (HisFeCh and His-FeChD347, respectively). However, the transition in activity was observed at higher substrate concentration than expected by metal binding to the His6-tag (1-3 molecules of Zn2+ would bind directly to the His6-tag). Therefore we assume that the presence of the His-tag affected the entry of substrate into the catalytic cleft [33,37,38], as well as the membrane-association properties of the enzyme. The N-terminal 166518-60-1 site domain of the catalytic cleft as well as the CAB-domain have been proposed to be involved in membrane binding of Synechocystis 6803 ferrochelatase in vivo [32]. Also, Zn2+ in solution can cause dimerization of Histags and therefore influence enzyme activity [39]. Removal of the His6-tag from His-FeCh or His-FeChD347, respectively, resulted in significant lower affinity for Zn2+ as judged by the higher binding constant KM. The opposite effect wa.Cystis 6803 was expressed in E. coli as inclusion bodies, unless coexpressed with the chaperones GroEL/GroES, while a construct depleted of this CAB motif, His-FeChD347, could be expressed fully soluble in E. coli. The use of standard purification protocols for purification of His-FeCh resulted either in truncation products or soluble aggregates, despite the usage of specialized E. coli strains (e.g. Rosetta2 (DE3)). The site of truncation was located at the Cterminal part of His-FeCh as revealed by immunoblotting and peptide mass fingerprinting (data not shown). We developed a protocol for the purification and refolding of recombinant ferrochelatase in order to circumvent these problems, and here we are able to show that recombinant full-length FeCh of Synechocystis 6803 is active as a monomer. In its monomeric state FeCh activity was dependent on the presence of potassium ions to stabilize the native protein structure through an apparent weak kosmotropic effect [30]. Recombinant FeCh activity was found to be insensitive to pH changes in the range of pH 6?, indicating additional regulation factors in vivo for this enzyme in photosynthetic organisms. Type I ferrochelatases are known to be regulated by the redox state of the cell [5], their activity was found to increase in response to environmental stresses, while type II activity is repressed under these conditions [5]. The temperature optimum of FeCh activity was at 30uC, coinciding with the typical growth temperature of Synechocystis 6803. At 37uC there was still appreciable activity of the enzyme, which then declined rapidly at higher temperatures (Fig. 4). The choice of detergent appears to be important for FeCh activity. Attachment to the photosynthetic membranes is required for type II ferrochelatases in vivo in order to pursue both uptake ofProto9 and release of heme [27]. b-DM forms oblate micelles mimicking a biological membrane, while CHAPS micelles have a prolate shape [34,35]. Therefore b-DM seems to be better suited for optimal activity of FeCh compared to CHAPS. The lag phase, resulting in a sigmoidal progress curve that was observed when measuring FeCh activity in the presence of CHAPS, could be abolished by pre-incubating the enzyme with metal ions before the start of the assay. The increased activity after the lag phase therefore was not due to a decreasing zinc pool. Enzyme kinetic plots revealed cooperativity of FeCh and FeChD347 regarding Zn2+, the substrate metal therefore might bind to peripheral sites of the enzymes [36,37]. This cooperativity was even more pronounced studying the His-tagged enzymes (HisFeCh and His-FeChD347, respectively). However, the transition in activity was observed at higher substrate concentration than expected by metal binding to the His6-tag (1-3 molecules of Zn2+ would bind directly to the His6-tag). Therefore we assume that the presence of the His-tag affected the entry of substrate into the catalytic cleft [33,37,38], as well as the membrane-association properties of the enzyme. The N-terminal domain of the catalytic cleft as well as the CAB-domain have been proposed to be involved in membrane binding of Synechocystis 6803 ferrochelatase in vivo [32]. Also, Zn2+ in solution can cause dimerization of Histags and therefore influence enzyme activity [39]. Removal of the His6-tag from His-FeCh or His-FeChD347, respectively, resulted in significant lower affinity for Zn2+ as judged by the higher binding constant KM. The opposite effect wa.
