MAGNESIUM CHELATASE - ASSOCIATION WITH RIBOSOMES AND MUTANT COMPLEMENTATION STUDIES IDENTIFY BARLEY SUBUNIT XANTHA-G AS A FUNCTIONAL COUNTERPART OF RHODOBACTER SUBUNIT BCHD

Magnesium chelatase catalyses the insertion of Mg2+ into protoporphyri n and is found exclusively in organisms which synthesise chlorophyll o r bacteriochlorophyll. Soluble protein preparations containing >10 mg protein/ml, obtained by gentle lysis of barley plastids and Rhodobacte r sphaeroplasts. inserted Mg into deuteroporphyrin IX in the presence of ATP at rates of 40 and 8 pmoles/mg protein per min, respectively. W ith barley extracts optimal activity was observed with 40 mM Mg2+. The activity was inhibited by micromolar concentrations of chloramphenico l. Mutations in each of three genetic loci, Xantha-f, -g and -h, in ba rley destroyed the activity. However, Mg-chelatase activity was recons tituted in vitro by combining pairwise the plastid stroma protein prep arations from non-leaky xantha-f, -g and -h mutants. This establishes that, as in Rhodobacter three proteins are required for the insertion of magnesium into protoporphyrin IX in barley. These three proteins, X antha-F, -G and -H, are referred to as Mg-chelatase subunits and they appear to exist separate from each other in vivo. Active preparations from barley and Rhodobacter yielded pellet and supernatant fractions u pon centrifugation for 90 min at 272 000 x g. The pellet and the super natant were inactive when assayed separately, but when they were combi ned activity was restored. Differential distribution of the Mg-chelata se subunits in the fractions was established by in vitro complementati on assays using stroma protein From the xantha-f; -g, and -h mutants. Xantha-G protein was confined to the pellet fraction, while Xantha-H w as confined to the supernatant. Reconstitution assays using purified r ecombinant BchH, BchI and partially purified BchD revealed that the pe llet fraction from Rhodobacter contained the BchD subunit. The pellet fractions from both barley and Rhodobacter contained ribosomes and had an A(260):A(280) ratio of 1.8. On sucrose density gradients both Xant ha-G and BchD subunits migrated with the plastid and bacterial ribosom al RNA, respectively.