Effect of iron on activity of soybean multi-subunit acetyl-coenzyme A carboxylase

Multi-subunit acetyl-coenzyme A carboxylase (MS-ACCase; EC 6.4.1.2) isolated from soybean chloroplasts is a labile enzyme that loses activity during purification. We found that incubating the chloroplast stromal fraction under anaerobic conditions or in the presence of 5 mM FeSO4 stimulated ACCase (acetyl-CoA --> malonyl-CoA) and carboxyltransferase (malonyl-CoA --> acetyl-CoA) activity. Fe-stimulation of activity was associated with Fe-59 binding to a stromal protein fraction. ACCase and carboxyltransferase activities measured in the stromal protein fraction containing bound Fe-59 were 2-fold and 6-fold greater, respectively, than the control (stromal fraction not pretreated with FeSO4). Superose 6 gel filtration chromatography indicated Fe-59 comigrated with stromal protein of approximately 180 kDa that exhibited carboxyltransferase activity, but lacked ACCase activity. Anion exchange (Mono-Q) chromatography of the Superose 6 fraction yielded a protein peak that was enriched in carboxyltransferase activity and contained protein-bound Fe-59. Denaturing gels of the Mono-Q fraction indicated that the 180-kDa protein was composed of a 56-kDa subunit that was bound by an antibody raised against a synthetic beta -carboxyltransferase (beta -CTase) peptide. Incubation of the Mono-Q carboxyltransferase fraction with increasing concentrations of iron at a fixed substrate concentration resulted in increased initial velocities that fit well to a single rectangular three parameter hyperbola (v = v(o) + V-max[FeSO4]/K-m + [FeSO4]) consistent with iron functioning as a bound activator of catalysis. UV/Vis spectroscopy of the partially purified fraction before and after iron incubation yielded spectra consistent with a protein-bound metal cluster. These results suggest that the beta -CTase subunit of MS-ACCase in soybean chloroplasts is an iron-containing enzyme, which may in part explain its labile nature.