Abstract
A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), catalyzed by dark-operative protochlorophyllide oxidoreductase (DPOR). DPOR is made of electron donor (BchL) and acceptor (BchNB) component proteins. BchNB is further composed of two subunits each of BchN and BchB arranged as an α2β2 heterotetramer with two active sites for substrate reduction. Such oligomeric architectures are found in several other electron transfer (ET) complexes, but how this architecture influences activity is unclear. Here, we describe allosteric communication between the two identical active sites in Rhodobacter sphaeroides BchNB that drives sequential and asymmetric ET. Pchlide binding to one BchNB active site initiates ET from the pre-reduced [4Fe-4S] cluster of BchNB, a process similar to the deficit spending mechanism observed in the structurally related nitrogenase complex. Pchlide binding in one active site is recognized in trans by an Asp-274 from the opposing half, which is positioned to serve as the initial proton donor. A D274A variant DPOR binds to two Pchlide molecules in the BchNB complex, but only one is bound productively, stalling Pchlide reduction in both active sites. A half-active complex combining one WT and one D274A monomer also stalled after one electron was transferred in the WT half. We propose that such sequential electron transfer in oligomeric enzymes serves as a regulatory mechanism to ensure binding and recognition of the correct substrate. The findings shed light on the functional advantages imparted by the oligomeric architecture found in many electron transfer enzymes.
Highlights
Many oligomeric enzymes that transfer electrons for catalysis or substrate reduction have two identical active sites and their subunits are arranged with a head-to-head or head-totail symmetry
An overarching question about these enzymes is centered on their conserved architectural complexity: Why are these enzymes assembled as two functional halves, and how do they cooperatively function during substrate reduction? Because two rounds of electron transfer (ET) are required for substrate reduction, multiple BchL binding and dissociation cycles occur at each BchNB half
A half-reactive dark-operative protochlorophyllide oxidoreductase (DPOR) complex is defective for substrate reduction
Summary
Many oligomeric enzymes that transfer electrons for catalysis or substrate reduction have two identical active sites and their subunits are arranged with a head-to-head or head-totail symmetry. We monitored Pchlide reduction activity of the dual-tagged WT and half-active BchNB complexes by mixing them with Pchlide, BchL, and ATP. Whereas WT BchNB carrying either affinity tag reduces Pchlide, the half-active BchNB is defective for Pchlide reduction (Fig. 2, a and b).
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