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, producing protochlorophyllide a and contributing substantially to the absorption properties of chlorophylls and bacteriochlorophylls. The O 2 -dependent cyclase is found in both oxygenic phototrophs and some purple bacteria. We overproduced the simplest form of the cyclase, AcsF, from Rubrivivax gelatinosus , in Escherichia coli . In biochemical assays the di-iron 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 yielded a turnover number of 0.9 min −1 , a Michaelis–Menten constant of 7.0 µM for MgPME and a dissociation constant for MgPME of 0.16 µM. Mass spectrometry identified 13 1 -hydroxy-MgPME and 13 1 -keto-MgPME as cyclase reaction intermediates, revealing the steps that form the isocyclic ring and completing the work originated by Sam Granick in 1950.