Kinetic and biophysical approaches to the assembly of magnesium chelatase

Magnesium chelatase catalyses the first committed step in chlorophyll biosynthesis, the insertion of Mg2+ into protoporphyrin-IX yielding magnesium protoporphyrin-IX; This enzyme stands at a branch-point in chlorophyll and heme biosynthesis. Mg2+ insertion is energetically unfavourable and is coupled with ATP hydrolysis. Magnesium chelatase from Synechocystis sp. PCC6803 contains three essential protein subunits ChlI, ChlD and ChlH. ChlI and ChlD belong to the AAA+ family of ATPases and form motor complexes that catalyse ATP hydrolysis which drives the Mg2+ insertion reaction occurring on ChlH. The number and arrangement of these protein subunits that represent the active chelatase are poorly understood. My work used a series of techniques to probe the complexes formed by the chelatase. Kinetic titrations show active complexes that catalyse either Mg2+ insertion or ATP
hydrolysis occur at two widely different levels of subunits, either 1:1 levels or at much
higher levels of ChlI over ChlD. ChlH influences these ChlID complexes, with high levels
of ChlH appearing to cause a reorganisation of higher order ChlID complexes as well
as causing a dramatic change in the ATPase activity of the system. An I6D6 complex has been demonstrated previously within the Rhodobacter capsulatus chelatase and a similar complex could be expected within the Synechocystis system. Sedimentation velocity analytical ultra centrifugation performed on the Synechocystis chelatase gives no evidence for this complex. A smaller complex is observed, favoured at high levels of ChlI, or in the presence of ChlH, this complex was proposed to represent an I5D complex or a similar arrangement. No ChlIDH complex was observed, although ChlH appears to influence the arrangement of smaller ChlID complexes. Modelling of the reaction cycle provides evidence for an isomerisation of ChlH preceding DIX substrate binding that may contribute to the observed lag phase prior to Mg2+
chelation.