How the O(2)-dependent Mg-protoporphyrin monomethyl ester cyclase forms the fifth ring of chlorophylls

Mg-protoporphyrin IX monomethyl ester (MgPME) cyclase catalyses the formation of the isocyclic ring, the hallmark of chlorins and bacteriochlorins, producing protochlorophyllide a and contributing significantly to the absorption properties of chlorophylls and bacteriochlorophylls. The O(2)-dependent cyclase is found in oxygenic phototrophs and in some purple bacteria. We overproduced the simplest form of the cyclase, AcsF from Rubrivivax gelatinosus, in Escherichia coli. In biochemical assays the diiron cluster within AcsF is reduced by ferredoxin furnished by NADPH and ferredoxin:NADP(+) reductase or by direct coupling to Photosystem I photochemistry, linking cyclase to the photosynthetic electron transport chain. Kinetic analyses yield a k (cat) of 0.9 min(-1), a K (M) of 7.0 µM for MgPME, and a K (d) for MgPME of 0.16 µM. Mass spectrometry identified 13(1)-hydroxy-MgPME and 13(1)-keto-MgPME as intermediates in the formation of the isocyclic ring, revealing the reaction chemistry that converts porphyrins to chlorins, and completing the work originated by Sam Granick in 1950.