Role of the distal phenylalanine 54 on the structure, stability, and ligand binding of Coprinus cinereus peroxidase

Resonance Raman and electronic absorption spectra obtained at various pH va lues for the Fe3+ form of distal F54 mutants of Coprinus cinereus peroxidas e are reported, together with the Fe2+ form and fluoride and imidazole addu cts at pH 6.0, 5.0, and 10.5, respectively. The distal phenylalanine residu e has been replaced by the small aliphatic residues glycine and valine and the hydrogen-bonding aromatic residues tyrosine and tryptophan (F54G, -V, - Y, and -W, respectively). These mutations resulted in transitions between f erric high-spin five-coordinate and six-coordinate forms, and caused a decr ease of the pK(a) of the alkaline transition together with a higher tendenc y for unfolding. The mutations also alter the ability of the proteins to bi nd fluoride in such a way that those that are six-coordinate at pH 5.0 bind more strongly than both wild-type CIP and F54Y which are five-coordinate a t this pH value. The data provide evidence that the architecture of the dis tal pocket of CTP is altered by the mutations. Direct evidence is provided that the distal phenylalanine plays an important role in controlling the co njugation between the vinyl double bonds and the porphyrin macrocycle, as i ndicated by the reorientation of the vinyl groups upon mutation of phenylal anine with the small aliphatic side chains of glycine and valine residues. Furthermore, it appears that the presence of the hydrogen-bonding tyrosine or tryptophan in the cavity increases the pK(a) of the distal histidine for protonation compared with that of wild-type CIP.