Structural and kinetic studies of imidazole binding to two members of the cytochrome c 6 family reveal an important role for a conserved heme pocket residue

The amino acid at position 51 in the cytochrome c 6 family is responsible for modulating over 100 mV of heme midpoint redox potential. As part of the present work, the X-ray structure of the imidazole adduct of the photosynthetic cytochrome c 6 Q51V variant from Phormidium laminosum has been determined. The structure reveals the axial Met ligand is dissociated from the heme iron but remains inside the heme pocket and the Ω-loop housing the Met ligand is stabilized through polar interactions with the imidazole and heme propionate-6. The latter is possible owing to a 180° rotation of both heme propionates upon imidazole binding. From equilibrium and kinetic studies, a Val residue at position 51 increases the stability of the Fe-S(Met) interaction and also affects the dynamics associated with imidazole binding. In this respect, the k obs for imidazole binding to Arabidopsis thaliana cytochrome c 6A, which has a Val at the position equivalent to position 51 in photosynthetic cytochrome c 6, was found to be independent of imidazole concentration, indicating that the binding process is limited by the Met dissociation rate constant (about 1 s -1). For the cytochrome c 6 Q51V variant, imidazole binding was suppressed in comparison with the wild-type protein and the V52Q variant of cytochrome c 6A was found to bind imidazole readily. We conclude that the residue type at position 51/52 in the cytochrome c 6 family is additionally responsible for tuning the stability of the heme iron-Met bond and the dynamic properties of the ferric protein fold associated with endogenous ligand binding. © 2011 SBIC.