Manipulating the covalent link between distal side tryptophan, tyrosine, and methionine in catalase-peroxidases: an electronic absorption and resonance Raman study.
Electronic absorption and resonance Raman spectroscopies have been applied to study the ferric and ferrous forms, and fluoride complexes of the Tyr249Phe and Met275Ile variants of the recombinant catalase-peroxidase (KatG) from the cyanobacterium Synechocystis PCC 6803. Both crystal structures and mass spectrometric analysis demonstrated that Tyr249 and Met275 are part of a novel KatG-specific covalent adduct including in addition a conserved tryptophan. Its role is not well established, but it has been shown to be essential for the catalase activity. In the present work we investigate the effect of mutation on the protein stability and ligand binding. The results clearly show that mutation weakens the heme binding to the protein, giving rise to a partial conversion from the 5-coordinate high spin of the wild-type protein to 6-coordinate low-spin heme. An internal ligand binds the heme iron on the distal side as a consequence of protein destabilization and partially prevents the binding of external ligand such as fluoride. The results are compared with those previously reported for the Trp122Ala and Trp122Phe variants.
Santoni
Elisa
E
Dipartimento di Chimica, Universita' di Firenze, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Florence, Italy.
Jakopitsch
Christa
C
Obinger
Christian
C
Smulevich
Giulietta
G
D002374:Catalase / Q000737:chemistry*; D018360:Crystallography, X-Ray; D000458:Cyanobacteria / Q000201:enzymology; D004583:Electrons; D005459:Fluorides / Q000737:chemistry; D019613:Haloarcula / Q000201:enzymology; D006418:Heme / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D008024:Ligands; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry*; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009195:Peroxidase / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman / Q000379:methods*; D014364:Tryptophan / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
D008024:Ligands; D011994:Recombinant Proteins; D014443:Tyrosine; D006418:Heme; D006859:Hydrogen; D014364:Tryptophan; D008715:Methionine; D007501:Iron; D002374:Catalase; D009195:Peroxidase; D005459:Fluorides