ATPase activity associated with the magnesium-protoporphyrin IX chelatase enzyme of Synechocystis PCC6803: evidence for ATP hydrolysis during Mg2+ insertion, and the MgATP-dependent interaction of the ChlI and ChlD subunits

Insertion of Mg2+ into protoporphyrin IX catalysed by the three-subunit enz yme magnesium-protoporphyrin IX chelatase (Mg chelatase) is thought to be a two-step reaction, consisting of activation followed by Mg2+ chelation. Th e activation step requires ATP and two of the subunits, ChlI and ChlD (I an d D respectively), and it has been speculated that this step results in the formation of an I-D-ATP complex. The subsequent step, in which Mg2+ is ins erted into protoporphyrin, also requires ATP and the third subunit, H, in a ddition to ATP-activated I-D complex. In the present study, we examine the interaction of the I and D subunits of the Mg chelatase from the cyanobacte rium Synechocystis PCC 6803. We demonstrate the purification of an I-D comp lex, and show that ATP and Mg2+ are absolute requirements for the formation of this complex, probably as MgATP. However, ATP may be replaced by the sl owly hydrolysable analogue, adenosine 5'-[gamma-thio]triphosphate, and, to a minor extent, by ADP and the non-hydrolysable ATP analogue, adenosine 5'- [beta,gamma-imido]triphosphate, all of which suggests that ATP hydrolysis i s not necessary for the formation of the ChlI-ChlD complex. A sensitive con tinuous assay was used to detect ATPase activity during Mg2+ chelation, and it was found that the maximum rate of ATP hydrolysis coincided with the ma ximum rate of Mg2+ insertion. The rate of ATP hydrolysis depended on factor s that determined the rate of Mg2+ chelation, such as increasing the concen tration of the H subunit and the concentration of protoporphyrin. Thus ATP hydrolysis has been identified as an absolute requirement for the chelation step. The I subunit possessed strong ATPase activity when assayed on its o wn: whereas the D subunit had no detectable activity, and when the I and D subunits were assayed in combination, the ATPase activity of the I subunit was repressed.