Identification of Precise Electrostatic Recognition Sites between Cytochrome c6 and the Photosystem I Subunit PsaF Using Mass Spectrometry

Frederik Sommer,Friedel Drepper,Wolfgang Haehnel,Michael Hippler

doi:10.1074/jbc.m607384200

Frederik Sommer, Friedel Drepper + Show 2 more

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https://doi.org/10.1074/jbc.m607384200

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Abstract

The reduction of the photo-oxidized special chlorophyll pair P700 of photosystem I (PSI) in the photosynthetic electron transport chain of eukaryotic organisms is facilitated by the soluble copper-containing protein plastocyanin (pc). In the absence of copper, pc is functionally replaced by the heme-containing protein cytochrome c6 (cyt c6) in the green alga Chlamydomonas reinhardtii. Binding and electron transfer between both donors and PSI follows a two-step mechanism that depends on electrostatic and hydrophobic recognition between the partners. Although the electrostatic and hydrophobic recognition sites on pc and PSI are well known, the precise electrostatic recognition site on cyt c6 is unknown. To specify the interaction sites on a molecular level, we cross-linked cyt c6 and PSI using a zero-length cross-linker and obtained a cross-linked complex competent in fast and efficient electron transfer. As shown previously, cyt c6 cross-links specifically with the PsaF subunit of PSI. Mass spectrometric analysis of tryptic peptides from the cross-linked product revealed specific interaction sites between residues Lys27 of PsaF and Glu69 of cyt c6 and between Lys23 of PsaF and Glu69/Glu70 of cyt c6. Using these new data, we present a molecular model of the intermolecular electron transfer complex between eukaryotic cyt c6 and PSI.

Highlights

In photosynthetic electron transfer, photosystem I (PSI)2 functions as an integral light-driven plastocyanin:ferredoxin oxidoreductase
Analysis of binding and electron transfer between the altered donors and the cyanobacterial PSI revealed that a positively charged amino acid located at the “northern face” of either pc or cyt c6 is crucial for the interaction with the reaction center (19 –21)
An equivalent positively charged exposed amino acid is present in cyt c6 from C. reinhardtii [22] but absent from pc of C. reinhardtii [23] and other eukaryotic pc structures

Summary

Introduction

Photosystem I (PSI) functions as an integral light-driven plastocyanin:ferredoxin oxidoreductase. Long range electrostatic attractions between basic patches of PsaF and acidic regions of pc and cyt c6 (8 –12) as well as the hydrophobic contact between the electron transfer site of the donors and PSI, including PsaATrp651 and PsaB-Trp627 in C. reinhardtii [9, 13], are required for stable electron transfer complex formation and efficient electron transfer. The change of either of the two PsaA or PsaB Trp residues to Phe abolished the formation of an intermolecular electron transfer complex between the altered PSI and pc, indicating that PsaATrp651 as well as PsaBTrp627 of loops i/j form the hydrophobic recognition site required for binding of pc to the core of PSI. Site-directed mutagenesis of eukaryotic pc has demonstrated that the “southern” conserved negatively charged patch and the northern hydrophobic flat site of pc are required for electrostatic attraction and hydrophobic contact between pc and PSI, respectively, to promote binding and efficient electron transfer between pc and PSI (8 –10,28)

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