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PreprintCC BY-NC-NDbioRxiv

A Binuclear Cluster Supports Functional Asymmetry in Dark-Operative Protochlorophyllide Reductase

  • K. Zhao
  • R. Kashyap
  • M. J. Abernathy
  • E. Antony
  • R. Sarangi1
  1. 1SLAC National Accelerator Laboratory
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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 2{beta}2 heterotetramer, and each heterodimeric half contains an active site and a [Fe4S4] 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 [Cu2S2] cluster. Each Cu is reduced and three-coordinate, with short Cu-S and Cu-Cu distances (2.26 and 2.62 &Aring). 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.

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Cite

BibTeXRIS
BibTeX
@misc{zhao2026binuclear,
  title = {{A Binuclear Cluster Supports Functional Asymmetry in Dark-Operative Protochlorophyllide Reductase}},
  author = {K. Zhao and R. Kashyap and M. J. Abernathy and E. Antony and R. Sarangi},
  howpublished = {bioRxiv},
  year = {2026},
  month = nov,
  doi = {10.64898/2026.11.03.763803},
  url = {https://www.biorxiv.org/content/10.64898/2026.11.03.763803v1},
  note = {CC BY-NC-ND},
}
RIS
TY  - UNPB
TI  - A Binuclear Cluster Supports Functional Asymmetry in Dark-Operative Protochlorophyllide Reductase
AU  - K. Zhao
AU  - R. Kashyap
AU  - M. J. Abernathy
AU  - E. Antony
AU  - R. Sarangi
PY  - 2026
DA  - 2026/11/03
PB  - bioRxiv
DO  - 10.64898/2026.11.03.763803
UR  - https://www.biorxiv.org/content/10.64898/2026.11.03.763803v1
AB  - 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 2{beta}2 heterotetramer, and each heterodimeric half contains an active site and a [Fe4S4] 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 [Cu2S2] cluster. Each Cu is reduced and three-coordinate, with short Cu-S and Cu-Cu distances (2.26 and 2.62 &Aring). 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.
ER  -