Substrate-binding model of the chlorophyll biosynthetic magnesium chelatase BchH subunit.

Photosynthetic organisms require chlorophyll and bacteriochlorophyll to harness light energy and transform water and carbon dioxide into carbohydrates and oxygen. The biosynthesis of these pigments is initiated by magnesium chelatase, an enzyme composed of BchI, BchD and BchH proteins, which catalyzes the insertion of Mg(2+) into protoporphyrin IX (Proto) to produce Mg-protoporphyrin IX. BchI and BchD form an ATP dependant AAA(+) complex that transiently interacts with the Proto-binding BchH subunit, at which point Mg(2+) is chelated. In this study, controlled proteolysis, electron microscopy of negatively stained specimens and single-particle 3D reconstruction have been used to probe the structure and substrate binding mechanism of the BchH subunit to a resolution of 25 A. The apo structure contains three major lobe-shaped domains connected at a single point, with additional densities at the tip of two lobes termed the thumb and finger. With the independent reconstruction of a substrate bound BchH complex (BchH-Proto) we observed a distinct conformational change in the thumb and finger subdomains. Prolonged proteolysis of native apo BchH produced a stable C-terminal fragment of 45 kDa, and Proto was shown to protect the full polypeptide from degradation. Fitting of a truncated BchH polypeptide reconstruction identified the N- and C-terminal domains. Our results show that the N- and C-terminal domains play crucial roles in the substrate-binding mechanism.