A single molecule approach to studying magnesium chelatase

The enzyme magnesium chelatase catalyses the insertion of magnesium into protoporphyrin IX to form magnesium protoporphyrin IX in the first committed step of chlorophyll biosynthesis. It comprises three different subunits known as ChlI, ChlD and ChlH. ChlI and ChlD interact in an ATP-dependent manner, forming a complex to which the porphyrin-carrying subunit ChlH binds. Magnesium insertion requires a large amount of energy provided by the concomitant ATP hydrolysis of the ChlI subunit. A wide range of experimental methods have been employed to determine the stoichiometry and size of the active ID complex, but no general consensus has as yet been reached. Single molecule subunit counting is another, complementary method with which to address the problem. By labelling the subunits of the ID complex with fluorescent molecules, it has been possible to monitor the numbers of subunits within individual complexes directly from the fluorescence intensity of the labels. This technique reveals that the form of the ID complex is dynamic and the number of ChlD subunits involved in the ID complex varies between 1 and 5. Previous work has investigated the ATPase activity and nucleotide binding kinetics of the ChlI subunit in ensemble measurements. This accessed the behaviour of the free enzyme species and determined that ChlI binds nucleotide according to the mechanism of conformational selection. Single molecule nucleotide binding experiments have probed the interaction of fluorescent nucleotide with the ChlI subunit directly. These provide a value for the off-rate binding constant of 6.0 ± 1.9 s-1 which suggests either that it is inappropriate to use the rapid equilibrium approximation when interpreting transient kinetic data for this subunit, or that the enzyme undergoes a second isomerization upon binding nucleotide.