Aerobic Barley Mg-protoporphyrin IX Monomethyl Ester Cyclase is Powered by Electrons from Ferredoxin

Chlorophyll is the light-harvesting molecule central to the process of photosynthesis.

Chlorophyll is synthesized through 15 enzymatic steps. Most of the reactions have been characterized

using recombinant proteins. One exception is the formation of the isocyclic E-ring characteristic

of chlorophylls. This reaction is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase

encoded by Xantha-l in barley ( Hordeum vulgare L.). The Xantha-l gene product (XanL) is a

membrane-bound diiron monooxygenase, which requires additional soluble and membrane-bound

components for its activity. XanL has so far been impossible to produce as an active recombinant protein

for in vitro assays, which is required for deeper biochemical and structural analyses. In the present

work, we performed cyclase assays with soluble and membrane-bound fractions of barley etioplasts.

Addition of antibodies raised against ferredoxin or ferredoxin-NADPH oxidoreductase (FNR) inhibited

assays, strongly suggesting that reducing electrons for the cyclase reaction involves ferredoxin and

FNR. We further developed a completely recombinant cyclase assay. Expression of active XanL

required co-expression with an additional protein, Ycf54. In vitro cyclase activity was obtained

with recombinant XanL in combination with ferredoxin and FNR. Our experiment demonstrates

that the cyclase is a ferredoxin-dependent enzyme. Ferredoxin is part of the photosynthetic

electron-transport chain, which suggests that the cyclase reaction might be connected to photosynthesis

under light conditions.