Methods for Nitrogenase-Like Dark Operative Protochlorophyllide Oxidoreductase

Abstract

Nitrogenase-like dark operative protochlorophyllide oxidoreductase (DPOR) is involved in the biosynthesis of chlorophylls and bacteriochlorophylls in gymnosperms, ferns, algae, and photosynthetic bacteria. During protochlorophyllide (Pchlide) reduction, the homodimeric subunit ChlL₂ of DPOR transfers electrons on the corresponding heterotetrameric catalytic subunit (ChlN/ChlB)₂. Although DPOR shares significant amino acid sequence homology to the nitrogenase system, only the initial catalytic steps of DPOR resemble nitrogenase catalysis. Investigation of the cyanobacterial DPOR from Prochlorococcus marinus indicated that subcomplex ChlL₂ is functioning as an ATP-dependent switch protein, triggering the transient interaction of ChlL₂ and (ChlN/ChlB)₂. This dynamic subunit interplay is responsible for the transfer of a single electron from the [4Fe–4S] cluster of ChlL₂ onto a second [4Fe–4S] cluster located on (ChlN/ChlB)₂. However, the second part of DPOR catalysis is unrelated to nitrogenase catalysis, since no molybdenum-containing cofactor or a P-cluster equivalent is employed. Instead, two consecutive electron transfer steps are mediated via the [4Fe–4S] cluster of (ChlN/ChlB)₂, resulting in the reduction of the conjugated ring system of the substrate molecule Pchlide (Figs. 5.1a and 5.2).