ATPase activity associated with the magnesium chelatase H-subunit of the chlorophyll biosynthetic pathway is an artefact.
Magnesium chelatase inserts Mg2+ into protoporphyrin IX and is the first unique enzyme of the chlorophyll biosynthetic pathway. It is a heterotrimeric enzyme, composed of I- (40 kDa), D- (70 kDa) and H- (140 kDa) subunits. The I- and D-proteins belong to the family of AAA+ (ATPases associated with various cellular activities), but only I-subunit hydrolyses ATP to ADP. The D-subunits provide a platform for the assembly of the I-subunits, which results in a two-tiered hexameric ring complex. However, the D-subunits are unstable in the chloroplast unless ATPase active I-subunits are present. The H-subunit binds protoporphyrin and is suggested to be the catalytic subunit. Previous studies have indicated that the H-subunit also has ATPase activity, which is in accordance with an earlier suggested two-stage mechanism of the reaction. In the present study, we demonstrate that gel filtration chromatography of affinity-purified Rhodobacter capsulatus H-subunit produced in Escherichia coli generates a high- and a low-molecular-mass fraction. Both fractions were dominated by the H-subunit, but the ATPase activity was only found in the high-molecular-mass fraction and magnesium chelatase activity was only associated with the low-molecular-mass fraction. We demonstrated that light converted monomeric low-molecular-mass H-subunit into high-molecular-mass aggregates. We conclude that ATP utilization by magnesium chelatase is solely connected to the I-subunit and suggest that a contaminating E. coli protein, which binds to aggregates of the H-subunit, caused the previously reported ATPase activity of the H-subunit.
Sirijovski
Nick
N
Department of Biochemistry, Lund University, Box 124, SE-22100 Lund, Sweden.
Olsson
Ulf
U
Lundqvist
Joakim
J
Al-Karadaghi
Salam
S
Willows
Robert D
RD
Hansson
Mats
M
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D016477:Artifacts; D001426:Bacterial Proteins / Q000378:metabolism; D002734:Chlorophyll / Q000096:biosynthesis*; D015964:Gene Expression Regulation, Bacterial; D008027:Light; D008190:Lyases / Q000378:metabolism*; D010944:Plants / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism; D011524:Protoporphyrins / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000378:metabolism
D001426:Bacterial Proteins; D021122:Protein Subunits; D011524:Protoporphyrins; D002734:Chlorophyll; C028025:protoporphyrin IX; D000251:Adenosine Triphosphatases; D008190:Lyases; C086788:magnesium chelatase