Modulation of heme redox potential in the cytochrome c6 family

Abstract

Cytochrome c6A is a unique dithio-cytochrome of green algae and plants. It has a very similar core structure to that of bacterial and algal cytochromes c6 but is unable to fulfill the same function of transferring electrons from cytochrome f to photosystem I. A key feature is that its heme midpoint potential is more than 200 mV below that of cytochrome c6 despite having His and Met as axial heme-iron ligands. To identify the molecular origins of the difference in potential, the structure of cytochrome c6 from the cyanobacterium Phormidium laminosum has been determined by X-ray crystallography and compared with the known structure of cytochrome c6A. One salient difference of the heme pockets is that a highly conserved Gln (Q51) in cytochrome c6 is replaced by Val (V52) in c6A. Using protein film voltammetry, we found that swapping these residues raised the c6A potential by +109 mV and decreased that of c6 by almost the same extent, -100 mV. X-ray crystallography of the V52Q protein showed that the Gln residue adopts the same configuration relative to the heme as in cytochrome c6 and we propose that this stereochemistry destabilizes the oxidized form of the heme. Consequently, replacement of Gln by Val was probably a key step in the evolution of cytochrome c6A from cytochrome c6, inhibiting reduction by the cytochrome b6f complex and facilitating establishment of a new function.

Figure 1. Three-dimensional structure of the homodimer of reduced P. laminosum cyt c ₆.

Chains A and B are colored green and salmon, respectively. The heme and axial ligands are in stick representation with the Fe as a red sphere. Each monomer is composed of four α-helices, three β-turns, and one γ-inverse turn. Helix 1 is distorted at C15 with residues 15–18 part of a 310 helix fragment. The N and C terminal helices are indicated for chain A.

Figure 2. Structural comparison of the heme binding sites.

(A) Sequence alignment between A. thaliana cyt c _6A and P. laminosum cyt c ₆ (sequence identity 34%). Residues whose side-chain atoms are within 5 Å of a heme atom are colored red to blue according to their hydrophobicity (blue = most polar, red = least polar), using the JalView alignment editor. (B and C) The heme binding pockets of reduced P. laminosum cyt c ₆ and reduced A. thaliana cyt c _6A, respectively. Water molecules are shown as small red spheres with the predominant polar interactions indicated by dashed lines; the larger spheres are the heme iron. The heme propionates are assigned as hp. A summary of the respective heme cavity polar interactions is given in Table S1.

Figure 3

(A) Cyclic voltammograms for cyt c _6A and cyt c ₆ adsorbed on a PGE electrode, A. thaliana cyt c _6A (black) and P. laminosum cyt c ₆ (gray), with their background corrected signals. The high potential redox couple is characteristic for protein-bound heme, whereas the low potential redox couple is from an unidentified contaminant (which is not visible on SDS gels and, hence, constitutes a small percentage of protein present but appears to react better with the electrode). Conditions: pH 7.1, scan rate 50 mV s^-1, 25 °C. (B) pH dependence of the midpoint potentials (E _m) at 25 °C. The data are fitted using the Nernst equation, E _m = E _acid – RT/F In[(1 + K _ox/a _H+)/(1 + Kred/a _H+)], where E _acid is the pH-independent value at low pH for pK _ox1 and pK _red1 (solid lines) and the pH-independent value at neutral pH for pK _ox2 (dashed lines).

Figure 4

(A) Part of a σ-weighted electron density map contoured at 1.0 σ for the oxidized V52Q-AA-c _6A variant structure of A. thaliana. The Gln52 and Met60 referred to in the text are indicated. (B) Stereoview of the heme regions of an overlay of reduced P. laminosum cyt c ₆ (light orange) and the oxidized V52Q-AA-c _6A variant of A. thaliana (pale cyan). Water molecules are shown as small spheres: red for P. laminosum cyt c 6 and blue for the V52Q-AA A. thaliana cyt c _6A variant. The iron atoms are shown as larger spheres: orange for P. laminosum and red for A. thaliana. Hydrogen bonding interactions are depicted by dashed lines; certain amino acids discussed in the text are labeled and shown as yellow sticks for A. thaliana or light orange sticks for P. laminosum.