Figure 1. Structure of Arabidopsis ferri-cytochrome c_6A I17A/G18A double mutant. (a) Structure in cartoon representation, with bound heme in stick representation and Fe ion as a red sphere. The characteristic LIP is highlighted in pink with the two cysteine residues forming a disulfide bond in yellow. (b) Electron density map to 1σ over the heme environment. The Fe coordinating Met and His are labelled, and the two covalently bound Cys are highlighted in yellow. Residues 17 and 18 where the Ala mutations were introduced are also labelled. The dotted line indicates a hydrogen bonding interaction between the N^δ atom of H20 and the main chain carbonyl of G24. (c) Structure-based sequence alignment of representative members of the land plant cytochrome c_6A family with the algal and cyanobacterial cytochrome c₆ family. The patterns of conservation are highlighted as follows: residues in red boxes are totally conserved, and sequence similarity based on the Blosum62 matrix in ClustalW⁵² is shown in red. The sequences are also annotated with the secondary structure of Arabidopsis cytochrome c_6A. The secondary structural elements are labelled: α1–α4 for the α-helices and η1 and η2 for the η-helices. The η2 helix is part of the LIP in the cytochrome c_6A family with the conserved Cys residues in yellow. The red stars indicate the conserved heme-binding motif, with the green triangle indicating a residue that may affect the reduction potential of the heme.

Figure 2. Spectroscopic analysis of Arabidopsis cytochrome c_6A (double mutant) crystals at 100 K. The key spectroscopic fingerprints of the ferric and ferrous oxidation states of the heme are observed. In the ferric spectrum, the arrow indicates the position of the 695 nm band, and the α and β- bands in the ferrous spectrum are labelled.

Figure 3. Superposition of the ferric and ferrous structures of cytochrome c_6A (double mutant). (a) The two structures (dark blue, ferric; light blue, ferrous) in cartoon representation. The heme is shown in stick representation with the Fe in red. The characteristic LIP of cytochrome c_6A is shown in pink (pink, ferric; light blue, ferrous), with the cysteine residues forming the disulfide bond in yellow. (b) Expanded view of the heme, showing the ligands Met and His. The colour-coding is the same as in (a). The conserved water molecule, found in both the ferric and ferrous oxidation states, is also indicated as a pink sphere.

Figure 4. Concentration dependence of k_obs for the reaction between wild-type cytochrome c_6A and pea plastocyanin at 300 K. The continuous line represents a linear regression, the slope being the second-order rate constant k₂. For reaction conditions see Materials and Methods.

Figure 5. Superposition of the structure of Arabidopsis ferri-cytochrome c_6A on that of ferro-cytochrome c₆ from the green alga Monoraphidium braunii (pdb code 1ctj). Blue, cytochrome c_6A; pink, cytochrome c₆.

Data processing and refinement statistics