Corless, Elliot I.Muhammad Saad Imran, SyedWatkins, Maxwell B.Bacik, John-PaulMattice, Jenna R.Patterson, AngelaDanyal, KaramatullahSoffe, MarkKitelinger, RobertSeefeldt, Lance C.Origanti, SofiaBennett, BrianBothner, BrianAndo, NozomiAntony, Edwin2022-09-082022-09-082021-01Corless, Elliot I., Syed Muhammad Saad Imran, Maxwell B. Watkins, John-Paul Bacik, Jenna R. Mattice, Angela Patterson, Karamatullah Danyal et al. "The flexible N-terminus of BchL autoinhibits activity through interaction with its [4Fe-4S] cluster and released upon ATP binding." Journal of Biological Chemistry 296 (2021).0021-9258https://scholarworks.montana.edu/handle/1/17097A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide to chlorophyllide, catalyzed by dark-operative protochlorophyllide oxidoreductase (DPOR). DPOR contains two [4Fe-4S]-containing component proteins (BchL and BchNB) that assemble upon ATP binding to BchL to coordinate electron transfer and protochlorophyllide reduction. But the precise nature of the ATP-induced conformational changes are poorly understood. We present a crystal structure of BchL in the nucleotide-free form where a conserved, flexible region in the N-terminus masks the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions in this region produce a hyper-active enzyme complex, suggesting a role for the N-terminus in auto-inhibition. Hydrogen deuterium exchange mass spectrometry shows that ATP-binding to BchL produces specific conformational changes leading to release of the flexible N-terminus from the docking interface. The release also promotes changes within the local environment surrounding the [4Fe-4S] cluster and promotes BchL complex formation with BchNB. A key patch of amino acids, Asp-Phe-Asp (the ‘DFD patch’), situated at the mouth of the BchL ATP-binding pocket promotes inter-subunit cross stabilization of the two subunits. A linked BchL dimer with one defective ATP-binding site does not support protochlorophyllide reduction, illustrating nucleotide binding to both subunits as a prerequisite for the inter-subunit cross stabilization. The masking of the [4Fe-4S] cluster by the flexible N-terminal region and the associated inhibition of activity is a novel mechanism of regulation in metalloproteins. Such mechanisms are possibly an adaptation to the anaerobic nature of eubacterial cells with poor tolerance for oxygen.en-UScc-byhttps://creativecommons.org/licenses/by/4.0/flexible BchL interation ATPArticle