ATPases and phosphate exchange activities in magnesium chelatase subunits of Rhodobacter sphaeroides

Abstract

Three separate proteins, BchD, BchH, and BchI, together with ATP, insert magnesium into protoporphyrin IX. An analysis of ATP utilization by the subunits revealed the following: BchH catalyzed ATP hydrolysis at the rate of 0.9 nmol per min per mg of protein. BchI and BchD, tested individually, had no ATPase activity but, when combined, hydrolyzed ATP at the rate of 117.9 nmol/min per mg of protein. Magnesium ions were required for the ATPase activities of both BchH and BchI+D, and these activities were inhibited 50% by 2 mM o-phenanthroline. BchI additionally catalyzed a phosphate exchange reaction from ATP and ADP. We conclude that ATP hydrolysis by BchI+D is required for an activation step in the magnesium chelatase reaction, whereas ATPase activity of BchH and the phosphate exchange activity of BchI participate in subsequent reactions leading to the insertion of Mg2+ into protoporphyrin IX.

Figure 1

Inhibition of ATPase and magnesium chelatase activities by o-phenanthroline and Co(III)–ATP–o-phenanthroline. (A) o-Phenanthroline inhibition of the ATPase activities of BchH (○) and BchD + BchI (•). (B) Inhibition of magnesium chelatase activity by o-phenanthroline (•) and Co(III)–ATP–o-phenanthroline (▵). The inhibitory effect of these compounds was tested in stopped assays containing (in μg of protein): BchD, 33; BchH, 210; and BchI, 90.

Figure 2

Effect of increasing the BchD concentration on the lag phase of the magnesium chelatase reaction. All of the ingredients in a total volume of 500 μl (20 mM Tricine-NaOH at pH 9.0, 1 mM DTT, 4 mM ATP, 20 mM creatine phosphate, 25 units of creatine kinase, 20 mM MgCl₂, 5 μM deuteroporphyrin IX, 0.72 mg of BchH, and 0.42 mg of BchI) except BchD were prewarmed to 30°C in a fluorometer cuvette. BchD was added, and increase in fluorescence emission at 580 nm was monitored continuously for 30 min with excitation light at 408 nm.

Figure 3

Effect of increasing the BchD concentration on the V_max. Magnesium chelatase activity was measured in continuous assays as described in Fig. 2. V_max is the maximum slope obtained after the lag phase.

Figure 4

The effect of NaF on the maximal velocity of the magnesium chelatase reaction. NaF effect was tested in continuous assays as described in Fig. 2.