Detection of the Photosystem I:Ferredoxin Complex by Backscattering Interferometry

Abstract

The dissociation constant K(d) of the photosystem I (PSI):ferredoxin complex has been measured by backscattering interferometry (BSI) with cyanobacterial PSI (350 kDa) and ferredoxin (10.5 kDa). The BSI signal, consisting of shifts for interference fringes resulting from a change in refractive index due to complex formation, was monitored as ferredoxin concentration was titrated. K(d) values of 0.14-0.38 microM were obtained with wild-type PSI whereas no complex was detectable with a PSI mutant containing a single mutation (R39Q) in the PsaE extrinsic subunit. These results are in quantitative agreement with previous functional determinations consisting in the detection of fast electron transfer within the complex. They provide evidence that the main contribution for the high affinity binding of ferredoxin to PSI is due to a single region of PsaE comprising arginine 39. They do not support the existence of a secondary binding site that could have escaped functional detection.

Interferometric Methods for Label-Free Molecular Interaction Studies

“…123 BSI found the affinity of the system to be 0.38 μ M, which is in good agreement with previous measurements of 0.2–0.6 μ M found using electron transfer (ET) kinetics. 124 The work also showed that binding occurred in a single-site manner, consistent with the current docking model.…”

Molecular interactions between photosystem I and ferredoxin: an integrated energy frustration and experimental model

“…Site-directed mutagenesis and chemical cross-linking studies have shown that the Lys34 residue of PsaC is potentially involved in docking Fd (Fischer et al, 1998). Backscattering interferometry data also indicate that the Arg39 residue of PsaE is also possibly implicated in binding Fd (Setif et al, 2010). These residues are both labeled in Figure 3.…”

“…In addition, research on Syn 6803 shows that after a short cross-linking time, Fd only cross-links to PsaD, and the proteolysis of the purified crosslinked product identified a covalent bond between Glu93 of Fd and Lys106 of the PsaD subunit (Lelong et al, 1994). Backscattering interferometry data also suggests that a single amino acid of PsaE is crucial for Fd binding to PSI (Setif et al, 2010). However, there has been no such work investigating this interaction in organisms that have high-resolution crystal structures, such as pea or Thermosynechococcus elongatus.…”

Mechanisms of resistance to paraquat in plants

“…With regard to the possibility of target site resistance, it is debatable whether paraquat has any specific binding site from which it accepts electrons. Paraquat is a better electrostatic fit to the negatively charged surface of the ferredoxin (Fd) which accepts electrons from PSI than it is to the site on PSI at which Fd binds and where paraquat would have to bind competitively with ferredoxin (where, for example, the PSI/Fd complex has a K d of ∼ 0.2 µM depending on species) . The measured midpoint redox potential of leaf ferredoxins (e.g.…”