In chlorophyll biosynthesis, the magnesium
chelatase enzyme complex catalyzes the insertion of a Mg2+
ion into protoporphyrin IX. Prior to this event, two of the three
subunits, the AAA+ proteins ChlI and ChlD, form a ChlID−
MgATP complex. We used microscale thermophoresis to
directly determine dissociation constants for the I-D subunits
from Synechocystis, and to show that the formation of a ChlID−
MgADP complex, mediated by the arginine finger and the
sensor II domain on ChlD, is necessary for the assembly of the
catalytically active ChlHID−MgATP complex. The N-terminal
AAA+ domain of ChlD is essential for complex formation, but
some stability is preserved in the absence of the C-terminal
integrin domain of ChlD, particularly if the intervening polyproline linker region is retained. Single molecule force spectroscopy
(SMFS) was used to determine the factors that stabilize formation of the ChlID−MgADP complex at the single molecule level;
ChlD was attached to an atomic force microscope (AFM) probe in two different orientations, and the ChlI subunits were
tethered to a silica surface; the probability of subunits interacting more than doubled in the presence of MgADP, and we show
that the N-terminal AAA+ domain of ChlD mediates this process, in agreement with the microscale thermophoresis data. Analysis
of the unbinding data revealed a most probable interaction force of around 109 pN for formation of single ChlID−MgADP
complexes. These experiments provide a quantitative basis for understanding the assembly and function of the Mg chelatase
complex