A Binuclear [Cu 2 S 2 ] Cluster Supports Functional Asymmetry in Dark-Operative Protochlorophyllide Reductase
Year: 2026
Type: preprint
Abstract: Many photosynthetic bacteria rely on the nitrogenase-like dark-operative protochlorophyllide oxidoreductase (DPOR) to sustain (bacterio)chlorophyll synthesis in the dark. DPOR couples ATP hydrolysis to electron transfer from a reducing component (L-protein) to a catalytic component (NB-protein) that reduces protochlorophyllide. NB-protein is an α 2 β 2 heterotetramer, and each heterodimeric half contains an active site and a [Fe 4 S 4 ] cluster. Although the two halves are identical, they react sequentially rather than in parallel. How the two halves communicate has remained unknown. Here we show, using Cu K-edge x-ray absorption spectroscopy and cryo-EM, that the NB-protein tetramer interface holds a binuclear, sulfur-bridged [Cu 2 S 2 ] cluster. Each Cu is reduced and three-coordinate, with short Cu-S and Cu-Cu distances (2.26 and 2.62 Å). Mutagenesis excludes the nearest cysteine as the bridging ligand, supporting inorganic sulfide as the most likely sulfur donor. To our knowledge, this is the first sulfide-bridged dicopper cluster identified in a protein. The cluster is conserved in three purple non-sulfur bacteria and connects the BchB C-terminal helices that contact opposing active sites. Loss of Cu abolishes DPOR activity and asymmetric L-protein binding without disturbing overall fold. We propose that the cluster relays substrate-binding information between active sites, suggesting a metal-mediated mechanism for cooperativity in nitrogenase-like enzymes.
Institutions: SLAC National Accelerator Laboratory, Saint Louis University
Language: English
Subfield: Biochemistry
Domain: Life Sciences
Open Access status: green
Funders: U.S. Department of Energy, Saint Louis University, National Institutes of Health, Office of Science, Center for Cellular Imaging, Washington University, National Institute of General Medical Sciences, Basic Energy Sciences, Biological and Environmental Research, SLAC National Accelerator Laboratory
Awards: G1229526393, G1286251239, G2109353418, G2371548206, G262532246, G2850949887, G303061748, G315621713, G3653110791, G4202012489, G4235786860, G4581610328, G495301720, G498139845, G5570207115, G6550344761, G7283024673, G7396808078, G7863156295, G8207748872, G8382000481, G8925327636, A Synchrotron Radiation Structural Biology Resource, G911061179
OpenAlex ID: W7225328536