Substrate recognition induces sequential electron transfer across subunits in the nitrogenase-like DPOR complex

Area of Science:

• Biochemistry
• Molecular Biology
• Photosynthesis Research

Background:

• Bacteriochlorophyll biosynthesis requires protochlorophyllide (Pchlide) reduction to chlorophyllide (Chlide).
• This reduction is catalyzed by dark-operative protochlorophyllide oxidoreductase (DPOR), a heterotetrameric complex (BchNB) with two active sites.
• The functional significance of such oligomeric architectures in electron transfer (ET) complexes remains largely unexplored.

Purpose of the Study:

• To investigate the allosteric communication and electron transfer mechanism in the BchNB complex from *Rhodobacter sphaeroides*.
• To elucidate how the oligomeric structure of DPOR influences its enzymatic activity and substrate reduction.
• To understand the role of specific residues, like Asp-274, in mediating inter-site communication and catalysis.

Main Methods:

• Biochemical characterization of wild-type and D274A variant DPOR complexes.
• Analysis of Pchlide binding and reduction kinetics in the BchNB heterotetramer.
• Construction and functional assessment of a half-active DPOR complex (one wild-type and one D274A monomer).

Main Results:

• DPOR exhibits sequential and asymmetric electron transfer between its two active sites, initiated by Pchlide binding.
• Asp-274 acts as a critical residue for inter-site communication, facilitating proton donation and productive substrate reduction.
• Mutating Asp-274 to Alanine (D274A) results in unproductive binding of two Pchlide molecules and stalled reduction.

Conclusions:

• Sequential electron transfer in oligomeric enzymes like DPOR serves as a regulatory mechanism for substrate binding and recognition.
• The oligomeric architecture of DPOR is crucial for its efficient and regulated function in bacteriochlorophyll biosynthesis.
• Findings provide insights into the functional advantages of oligomeric structures in diverse electron transfer enzymes.