Insights into the binding behavior of native and non-native cytochromes to photosystem I from Thermosynechococcus elongatus

Abstract

The binding of photosystem I (PS I) from Thermosynechococcus elongatus to the native cytochrome (cyt) c₆ and cyt c from horse heart (cyt c_HH) 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 c_HH 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 c_HH besides one specific binding site close to P₇₀₀ Binding to PS I was found to be the same for reduced and oxidized cyt c_HH Based on this information, suitable conditions for cocrystallization of cyt c_HH with PS I were found, resulting in crystals with a PS I:cyt c_HH ratio of 1:1. A crystal structure at 3.4-Å resolution was obtained, but cyt c_HH 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.

Keywords: complex; crystallography; cytochrome c; docking; photosynthesis; photosystem I.

Figure 1.

Oxygen reduction rates of PS I with 16 μm cyt c_HH (top) and cyt c₆ (bottom) at pH 8 (left) and pH 6 (right) as a function of ionic strength. Monovalent (NaCl; black) and divalent (MgCl₂; red) cations are depicted as circles and squares, respectively. For cyt c₆, pH 8 (bottom, left) differences between the applied salts become prominent. Therefore, a further differentiation of the salts is shown: NaCl (black), Na₂SO₄ (yellow), NH₄Cl (blue), MgCl₂ (red), CaCl₂ (gray), and MgSO₄ (cyan). NaCl, MgCl₂, and CaCl₂ are connected by a line in their corresponding color. All measurements were performed in either 25 mm Tricine-NaOH, pH 8, or 5 mm MES-NaOH, pH 6, with 2 mm ascorbic acid and 300 μm methyl viologen at 20 °C. The concentration of buffer ions and counterions, which contribute to the ionic strength, was calculated using the Henderson–Hasselbalch equation with a pK_a of 8.2 and 6.2 for Tricine and MES buffers, respectively. Standard deviations (error bars) were determined from three to nine independent measurements.

Figure 2.

Isothermal titration calorimetry of PS I with cyt c_HH. A, thermogram for exemplary background measurements (top) and oxidized (middle) and reduced (bottom) proteins. B, integrated heats of titrations after background subtraction in the presence (red) or absence (black) of 5 mm ascorbate. High cyt c_HH:P₇₀₀ ratios are omitted for a better overview. Fits (top) and residuals (bottom) are shown for one set of binding sites with n = 1.0 (dashed line) and for one set of binding sites with n = 1.5 (solid line). Parameters obtained from the models are shown in Table 2. Measurements were performed at 20 °C in 25 mm Tricine buffer, pH 8.0, with 25 mm NaCl and 0.02% DDM. Each titration step consisted of a 5-μl injected volume from 1 mm cyt c_HH.

Figure 3.

Isothermal titration calorimetry of PS I with cyt c₆. A, thermogram for exemplary background measurements (top, red) and oxidized (middle) and reduced (bottom) proteins. B, integrated heats of titrations after background subtraction in the presence (red; reduced) or absence (black; oxidized) of 5 mm ascorbate. The fit of the data for reduced cyt c₆ is shown for one set of binding sites with n = 1.0. Parameters obtained from the model are shown in Table 2. After substraction of the heat of dilution, the data for oxidized cyt c₆ converge to negative values at high cyt c₆:PS I ratio (−0.1 kcal/mol; not shown) and are thus not analyzed by a model. Measurements were performed at 20 °C in 25 mm Tricine-NaOH, pH 8.0, with 25 mm NaCl and 0.02% DDM. Each titration step consisted of a 5-μl injected volume from 1 mm cyt c₆.

Figure 4.

Molecular docking simulation of monomeric PS I with cyt c_HH (left) and cyt c₆ (right). Each sphere represents the position of a docked cyt c. The binding energy, calculated by pyDock, is highlighted by a color code. Docking states with less than −20 kcal/mol are highlighted by an increased sphere size.

Figure 5.

Potential cyt c_HH (top)– and cyt c₆ (bottom)–binding site of PS I. Shown are the docking sites that most likely resemble the specific cyt c–binding site of PS I. The heme group (red) of cyt c_HH and cyt c₆ points toward the luminal tryptophan residues Trp-A655 and Trp-B631 (blue) and P₇₀₀ (green) of PS I. The distances between the heme groups and the closest tryptophan are highlighted by a black dotted line. Cyt c₆ does not interact with PsaF (purple) but is close to the luminal loop of PsaA (yellow). The carboxyl group of Glu-34 from cyt c₆ is at a distance of 7.4 Å from the carboxyl group of Asp-628 from PsaA (gray dotted line).

Figure 6.

Superposition of the potential cyt c₆–binding site to the known cyt c₂–binding site of the bRC from R. sphaeroides (Protein Data Bank code 1l9b (34)). The superposition was achieved by aligning the heme groups. The right view is rotated by 90° with respect to the left view. The distances of the heme iron from cyt c₆ to the Mg²⁺ ions of P₇₀₀ are 21.4 and 21.3 Å, respectively. These distances are identical to the distances between the heme iron of cyt c₂ and the Mg²⁺ ions of P₈₇₀ (pink) from bRC (21.3 and 21.2 Å, respectively).