Figure 5. Effects of cysteine and tyrosine on in vitro HydA1 activation.

Maturase extracts (final concentrations of 50–60% vol·vol⁻¹) were reconstituted with Fe⁺² and S⁻² for 60 min, and then pre-treated with SAM and amino acids for 60 min prior to addition of apoHydA1 (black bars) or apoHydA1^recon (red bars). No additional molecules were added with HydA1 apoprotein (3.6–4.6 µM). Final concentrations of exogenous molecules were as follows: 1 mM Fe⁺², 1 mM S⁻², 2 mM SAM, 2 mM cysteine, 2 mM tyrosine, and 2 mM methionine. ( Fig. 5A ) Hydrogenase activities were measured after 8–9 hr of incubation. Data are the average for n = 2 to 5 independent determinations ± SEM. ApoHydA1^recon was only tested for mixtures with tyrosine and with cysteine plus tyrosine. Addition of methionine did not enhance hydrogenase activities for all four conditions (data not shown). ( Fig. 5B ) Reaction mixtures included as-isolated apoHydA1, Fe⁺², S⁻², SAM, and the following amino acids added as described above: cysteine (▪); tyrosine (×); cysteine and tyrosine (○). Data are the average for n = 2 independent determinations. Standard errors were less than 11% for all data.