Figure 5.

SDS-PAGE analysis of protein compositions of in vitro assays at different maturation steps. A, in vitro processing of hydrogenase-2 catalytic subunit HybC. Lane 1, reaction mix contained HybC, GDE complex, HypE, and HypF in stoichiometric amounts (2 μm each) and AM. Lane 2, the reaction was started by the addition of endopeptidase HybD in catalytic amounts, and immediately an aliquot of the reaction mix (∼25 μg) was analyzed (t₀). No HybC^−15AA was detected. Lanes 3–5, aliquots of the reaction mix were analyzed after 10, 20, and 30 min of incubation at room temperature, respectively (t₁₀, t₂₀, and t₃₀). HybC^−15AA formation was observed over time. After 20 min, no additional processing of HybC was observed. Lane 6, isolation of both unprocessed ^StrepHybC and processed ^StrepHybC^−15AA from the reaction mix by Strep-Tactin affinity chromatography. B, formation of HybC–HybO holoenzyme. Lane 1, ^HisHybO-enriched fraction. Lane 2, the ^StrepHybC and ^StrepHybC^−15AA fraction isolated from reaction mix was supplemented with the ^HisHybO-enriched fraction. Lane 3, the ^StrepHybC–^HisHybO heterocomplex was isolated by a histidine affinity column. Only the processed large subunit (HybC^−15AA) can form a heterodimer with the small subunit HybO.