Abstract
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.