Current limits of structural biology: The transient interaction between cytochrome c 6 and photosystem I

Abstract

Trimeric photosystem I from the cyanobacterium Thermosynechococcus elongatus (TePSI) is an intrinsic membrane protein, which converts solar energy into electrical energy by oxidizing the soluble redox mediator cytochrome c ₆ (Cyt c ₆ ) and reducing ferredoxin. Here, we use cryo-electron microscopy and small angle neutron scattering (SANS) to characterize the transient binding of Cyt c ₆ to TePSI. The structure of TePSI cross-linked to Cyt c ₆ was solved at a resolution of 2.9 Å and shows additional cofactors as well as side chain density for 84% of the peptide chain of subunit PsaK, revealing a hydrophobic, membrane intrinsic loop that enables binding of associated proteins. Due to the poor binding specificity, Cyt c ₆ could not be localized with certainty in our cryo-EM analysis. SANS measurements confirm that Cyt c ₆ does not bind to TePSI at protein concentrations comparable to those for cross-linking. However, SANS data indicate a complex formation between TePSI and the non-native mitochondrial cytochrome from horse heart (Cyt c _HH ). Our study pinpoints the difficulty of identifying very small binding partners (less than 5% of the overall size) in EM structures when binding affinities are poor. We relate our results to well resolved co-structures with known binding affinities and recommend confirmatory methods for complexes with K _M values higher than 20 μM.

Keywords: Cryo EM; Cytochrome c6, electron transfer; Electron transfer; Photo-biotechnology; Photosystem I; Small angle neutron scattering; Thermosynechococcus elongatus.

Graphical abstract

Fig. 1

Cryo-EM structure of the monomeric TePSI crosslinked to Cyt c₆ derived from the symmetry expanded dataset shown from the luminal side (left) and from along the membrane plane (right). The inset on the far left shows the position of the monomer in the PSI trimer as seen from the luminal side.

Fig. 2

Focused 3D classifications on the PSI monomer map using differently placed and sized soft masks. The model in the centre depicts monomeric PSI bound to Cyt c₆ with mask locations on luminal (A, teal mask and B, magenta mask) and stroma sides (C, gold mask and D, light blue mask). The densities derived from 3D classifications of the symmetry expanded monomer dataset are shown colour coded by mask used and contoured at 5 sigma (mesh) and 20 sigma (surface). The class occupancy for each class of each focussed classification is given.

Fig. 3

The structure of PsaK loop 32–55. A, View onto cytoplasmic side of the model with symmetry expanded monomeric EM map displayed as blue mesh (left) and view from membrane onto the same section of PsaK with modelled palmitoyloleoyl-phosphatidylglycerid- (POPG)-membrane (right). The PIALPAL-patch 44–50 is highlighted in green, and the salt bridge forming residues Arg31 and Glu52 are coloured blue and red, respectively. POPG membrane was simulated using CHARMM membrane builder (Jo et al., 2008). The map is plotted at a B-factor of −173 Å ². B, Model of trimer-trimer interface of T. elongatus thylakoid membrane. Hexagonal packing of PSI trimers (left) is based on the AFM images as shown in (MacGregor-Chatwin et al., 2017). Close up of the interface between two neighbouring trimers (right). The subunits at the interface are PsaF (orange), PsaJ (magenta) and PsaK (yellow). The PIALPAL-patch 44–50 of PsaK is highlighted in green. PsaK Glu52 (red), Arg31 and PsaF Lys108 (blue) are shown in stick representation.

Fig. 4

SANS measurements of TePSI in the presence of Cyt c_HH or Cyt c₆. A, SANS data of trimeric TePSI (black line) and of TePSI-Cyt c_HH complexes (red line) obtained at a contrast of 5% D₂O. B, Magnification of the SANS data shown in Panel A for the q-region, where a different signal is observed for trimeric TePSI (black line) and TePSI-Cyt c_HH complexes (red line), respectively. C, Pair distance distribution functions P(r) obtained from the SANS data of trimeric TePSI (black line) and of TePSI-Cyt c_HH complexes (red line), respectively. For comparison, we also present the P(r) function (green line) calculated from the crystal structure of TePSI (pdb code 1jb0; Jordan et al., 2001). D, SANS data of trimeric TePSI (black line) and of a mixture of TePSI-Cyt c₆ complexes (orange line) obtained at a contrast of 5% D₂O. E, Comparison of the structure of the TePSI-Cyt c_HH complex reconstructed from the SANS data using the ATSAS routine (light blue spheres) with a crystal structure of the PSI-cytochrome complex taken from (Kölsch et al., 2018).