Affinity and activity of non-native quinones at the QB site of bacterial photosynthetic reaction centers

Abstract

Purple, photosynthetic reaction centers (RCs) from Rb. sphaeroides bacteria use UQ10 as primary (QA) and secondary (QB) electron acceptors. Many quinones reconstitute QA function, while few will act as QB. Nine quinones were tested for their ability to bind and reconstitute QA and QB function. Only ubiquinone (UQ) reconstitutes both QA and QB function in the same protein. The affinities of the non-native quinones for the QB site were determined by a competitive inhibition assay. The affinities of benzoquinones (BQ), napthoquinone (NQ) and 2-methyl-NQ for the QB site are 7±3 times weaker than for the QA site. However, di-ortho substituted NQs and anthraquinone bind tightly to the QA site (Kd ≤200 nM) and ≥1000 times more weakly to the QB site, perhaps setting a limit on the size of the site. With a low potential electron donor (2-methyl, 3-dimethylamino-1,4-Napthoquinone (Me-diMeAm-NQ)) at QA, QB reduction is 260 meV more favorable than with UQ as QA. Electron transfer from Me-diMeAm-NQ at the QA site to NQ at the QB site can be detected. In the QB site the NQ semiquinone is estimated to be ≈ 60–100 meV higher in energy than the UQ semiquinone, while in the QA site the semiquinone energy level is similar or lower with NQ than with UQ. Thus, the NQ semiquinone is more stable in the QA than QB site. In contrast, the native UQ semiquinone is ≈ 60 meV lower in energy in the QB than the QA site, stabilizing forward electron transfer from QA to QB.

The 2-Methoxy Group Orientation Regulates the Redox Potential Difference between the Primary (QA) and Secondary (QB) Quinones of Type II Bacterial Photosynthetic Reaction Centers

“…Only an out-of-plane-oriented 2-methoxy group can elevate the electron affinity of Q B sufficiently to overcome a net unfavorable site solvation effect and render electron transfer from Q A thermodynamically favorable. Strong corroboration comes from a recent experimental study in which naphthoquinones were tested for Q B activity . These quinones, lacking methoxy groups, were found to exhibit Q B redox potentials 60–100 mV more negative than expected by comparison with the native ubiquinone and were only reducible when a low-potential quinone was present in the Q A site.…”

“…Strong corroboration comes from a recent experimental study in which naphthoquinones were tested for Q B activity. 14 These quinones, lacking methoxy groups, were found to exhibit Q B redox potentials 60− 100 mV more negative than expected by comparison with the native ubiquinone and were only reducible when a lowpotential quinone was present in the Q A site.…”

Molecular mechanisms for generating transmembrane proton gradients

“…There has been significant computational analysis of how the protein accomplishes this tuning [61, 105, 134, 135, 154, 170, 171]. Interestingly, no other pair of quinones can substitute for ubiquinone at both Q A and Q B , indicating the protein interacts with this specific compound in a special way [172–175]. …”

Introduction of the Menaquinone Biosynthetic Pathway into Rhodobacter sphaeroides and de Novo Synthesis of Menaquinone for Incorporation into Heterologously Expressed Integral Membrane Proteins

“…Studies of quinone binding or in vitro replacement in the RC determined that the environment of the binding pocket created by the surrounding amino acid residues is a major factor in the selectivity for a specific type of quinone. − In RCs that contain MK in the Q A site and UQ in the Q B pocket the driving force for electron transfer from Q A to Q B may derive to a large extent from the intrinsically more positive electrochemical midpoint potential of UQ than MK (about 100 mV) . However, the binding pocket protein matrix and side groups of the quinone headgroup function to tune the midpoint potential of the quinones such that the forward electron transfer driving force is thermodynamically favorable, in cases for which the same molecule is used in both pockets. − In other words, even though UQ has an intrinsic midpoint potential in solution, when UQ is assembled in the Q A and Q B pockets, the midpoint potentials differ due to the orientation of substituents on the quinone headgroup and interactions with the surrounding protein environment.…”