Before the start of the assay. (A) 50 nM co-expressed His-FeCh with

Before the start of the assay. (A) 50 nM co-expressed His-FeCh with 1 mM Zn2+, 0.5 mM Proto9 and pigment mix consisting of 0.5 mM Chl and 0.23 mM carotenoids. (B) 30 nM FeChD347, 5 mM Zn, 0.5 mM Proto9 and pigment mix consisting of 1.1 mM Chl and 0.5 mM carotenoids. doi:10.1371/journal.pone.0055569.gInfluence of Temperature, pH and Buffer Composition on Enzyme ActivityMaximal Defactinib site activity was reached when His-FeCh was in its monomeric form, in agreement with ferrochelatase purified from Synechocystis 6803 [32], however, a mixure of monomers and oligomers was comparable in activity (Fig. 2D). Buffer composition was chosen for negligible binding of divalent metal ions; Tris proved to be a good choice (Fig. 3) and also is commonly used in similar studies. The buffers 2-(N-morpholino)ethanesulfonic acid (MES) and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (Hepes) were suitable as well, however, borate as well as Hepes impaired His-FeCh activity at elevated temperatures (37uC) (data not shown). The choice of detergent was important for enzyme activity (Fig. 3), with the usage of CHAPS instead of b-DM leading to a decreased His-FeCh activity. Also the presence of cations was observed as being important for high activity (Fig. 3). Removal of potassium ions reduced the His-FeCh activity considerably while the addition of manganese ions to the standard buffer slightly increased its activity (Fig. 3). Recombinant His-FeCh activity was insensitive to pH variations in the range of pH 6 to 9 (Fig. 4A) and its temperature optimum was at or around 30uC (Fig. 4B), corresponding to the normal growth temperature of Synechocystis 6803. Based on the refolding results and activity data, an assay buffer composed of 50 mM Tris-HCl, pH 8, 0.1 M NaCl, 0.5 M KCl, 20 (v/v) glycerol, 1 mM b-DM and 0.025 (v/v) Tween-80 Table 3. Enzyme kinetic parameters for ferrochelatases from other species.kcat/min-1 0.19?.25 mM (Zn ) 0.09 mM 1.960.3 mM (Fe2+) 1.460.2 mM2+(maintaining solubility of Proto9) was used in the following measurements. Enzyme activity was Defactinib web typically measured at 30uC.Enzyme KineticsIt is well known that ferrochelatases can accept different divalent metal ions and various porphyrin substrates [33]. To be able to compare our data on the recombinant type II FeCh of Synechocystis 6803 with activity data observed for orthologues or other ferrochelatases, Zn2+ and the natural Proto9 were chosen as substrates. Full-length refolded His-FeCh (Fig. 5 A, C) and truncated His-FeChD347 (Fig. 5 B, D) exhibited very similar KM for these substrates. Interestingly both enzymes displayed strong cooperativity regarding the Zn2+ substrate (Fig. 5 A-B and Table 1). The midpoint transition varied slightly from batch to batch, but always remained in the sub-mM range (Table 1). The progress curves (measuring product formation over time) of HisFeCh and His-FeChD347 had hyperbolic shape when the standard assay was used (as in Fig. 5), but when the detergent bDM was exchanged against 10 mM Chaps, an initial lag phase of the enzymes became obvious, resulting in a sigmoidal shape. This lag phase could be avoided if the enzymes were pre-incubated with substrate metal for 10 min prior to the assay start (not shown). Removal of the His6-tag lowered the KM of FeCh for Proto9, while the KM for Zn2+ was increased and the cooperative effect was less pronounced (Fig. 6 and Table 2). For FeChD347 the turnover number kcat was moderately affected by removal of the His6-tag, while in contr.Before the start of the assay. (A) 50 nM co-expressed His-FeCh with 1 mM Zn2+, 0.5 mM Proto9 and pigment mix consisting of 0.5 mM Chl and 0.23 mM carotenoids. (B) 30 nM FeChD347, 5 mM Zn, 0.5 mM Proto9 and pigment mix consisting of 1.1 mM Chl and 0.5 mM carotenoids. doi:10.1371/journal.pone.0055569.gInfluence of Temperature, pH and Buffer Composition on Enzyme ActivityMaximal activity was reached when His-FeCh was in its monomeric form, in agreement with ferrochelatase purified from Synechocystis 6803 [32], however, a mixure of monomers and oligomers was comparable in activity (Fig. 2D). Buffer composition was chosen for negligible binding of divalent metal ions; Tris proved to be a good choice (Fig. 3) and also is commonly used in similar studies. The buffers 2-(N-morpholino)ethanesulfonic acid (MES) and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (Hepes) were suitable as well, however, borate as well as Hepes impaired His-FeCh activity at elevated temperatures (37uC) (data not shown). The choice of detergent was important for enzyme activity (Fig. 3), with the usage of CHAPS instead of b-DM leading to a decreased His-FeCh activity. Also the presence of cations was observed as being important for high activity (Fig. 3). Removal of potassium ions reduced the His-FeCh activity considerably while the addition of manganese ions to the standard buffer slightly increased its activity (Fig. 3). Recombinant His-FeCh activity was insensitive to pH variations in the range of pH 6 to 9 (Fig. 4A) and its temperature optimum was at or around 30uC (Fig. 4B), corresponding to the normal growth temperature of Synechocystis 6803. Based on the refolding results and activity data, an assay buffer composed of 50 mM Tris-HCl, pH 8, 0.1 M NaCl, 0.5 M KCl, 20 (v/v) glycerol, 1 mM b-DM and 0.025 (v/v) Tween-80 Table 3. Enzyme kinetic parameters for ferrochelatases from other species.kcat/min-1 0.19?.25 mM (Zn ) 0.09 mM 1.960.3 mM (Fe2+) 1.460.2 mM2+(maintaining solubility of Proto9) was used in the following measurements. Enzyme activity was typically measured at 30uC.Enzyme KineticsIt is well known that ferrochelatases can accept different divalent metal ions and various porphyrin substrates [33]. To be able to compare our data on the recombinant type II FeCh of Synechocystis 6803 with activity data observed for orthologues or other ferrochelatases, Zn2+ and the natural Proto9 were chosen as substrates. Full-length refolded His-FeCh (Fig. 5 A, C) and truncated His-FeChD347 (Fig. 5 B, D) exhibited very similar KM for these substrates. Interestingly both enzymes displayed strong cooperativity regarding the Zn2+ substrate (Fig. 5 A-B and Table 1). The midpoint transition varied slightly from batch to batch, but always remained in the sub-mM range (Table 1). The progress curves (measuring product formation over time) of HisFeCh and His-FeChD347 had hyperbolic shape when the standard assay was used (as in Fig. 5), but when the detergent bDM was exchanged against 10 mM Chaps, an initial lag phase of the enzymes became obvious, resulting in a sigmoidal shape. This lag phase could be avoided if the enzymes were pre-incubated with substrate metal for 10 min prior to the assay start (not shown). Removal of the His6-tag lowered the KM of FeCh for Proto9, while the KM for Zn2+ was increased and the cooperative effect was less pronounced (Fig. 6 and Table 2). For FeChD347 the turnover number kcat was moderately affected by removal of the His6-tag, while in contr.