Abstract
The binding of photosystem I (PS I) from Thermosynechococcus elongatus to the native cytochrome (cyt) c6 and cyt c from horse heart (cyt cHH) was analyzed by oxygen consumption measurements, isothermal titration calorimetry (ITC), and rigid body docking combined with electrostatic computations of binding energies. Although PS I has a higher affinity for cyt cHH than for cyt c6, the influence of ionic strength and pH on binding is different in the two cases. ITC and theoretical computations revealed the existence of unspecific binding sites for cyt cHH besides one specific binding site close to P700 Binding to PS I was found to be the same for reduced and oxidized cyt cHH Based on this information, suitable conditions for cocrystallization of cyt cHH with PS I were found, resulting in crystals with a PS I:cyt cHH ratio of 1:1. A crystal structure at 3.4-Å resolution was obtained, but cyt cHH cannot be identified in the electron density map because of unspecific binding sites and/or high flexibility at the specific binding site. Modeling the binding of cyt c6 to PS I revealed a specific binding site where the distance and orientation of cyt c6 relative to P700 are comparable with cyt c2 from purple bacteria relative to P870 This work provides new insights into the binding modes of different cytochromes to PS I, thus facilitating steps toward solving the PS I-cyt c costructure and a more detailed understanding of natural electron transport processes.
Highlights
The binding of photosystem I (PS I) from Thermosynechococcus elongatus to the native cytochrome c6 and cyt c from horse heart was analyzed by oxygen consumption measurements, isothermal titration calorimetry (ITC), and rigid body docking combined with electrostatic computations of binding energies
In this study we focused on an investigation of the binding properties of PS I from T. elongatus with cyt c from horse heart (cyt cHH) and cyt c6 under a variety of buffer conditions for elucidating the binding site
By using isothermal titration calorimetry and rigid body docking, we revealed that there is more than one cyt cHH– binding site at PS I under low ionic strength
Summary
The binding of photosystem I (PS I) from Thermosynechococcus elongatus to the native cytochrome (cyt) c6 and cyt c from horse heart (cyt cHH) was analyzed by oxygen consumption measurements, isothermal titration calorimetry (ITC), and rigid body docking combined with electrostatic computations of binding energies. Mutagenesis studies indicated that optimal binding of both electron mediators for electron transfer to P700ϩ occurs at a hydrophobic binding site, which is formed by two parallel tryptophan residues, Trp-655 from PsaA (Trp-A655) and Trp-632 from PsaB (Trp-B632) [10] Besides this hydrophobic site, a second binding site exists in plant and algal PS I that is based on electrostatic interactions due to positively charged side chains of PsaF. After binding to the charged site, PC reorients itself to bind to the hydrophobic area and form the active complex [11, 12] This binding model is based c; DDM, dodecyl -D-maltoside; DLS, dynamic light scattering; ITC, isothermal titration calorimetry; RC, reaction center; Trp-A655 and Trp-B632, tryptophan 655 and 632 from PsaA and PsaB, respectively; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PC, plastocyanin; mS, millisiemens
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