The effects of ligand exchange and mobility on the peroxidase activity of a bacterial cytochrome c upon unfolding.
The effect on the heme environment upon unfolding Paracoccus versutus ferricytochrome c-550 and two site-directed variants, K99E and H118Q, has been assessed through a combination of peroxidase activity increase and one-dimensional NMR spectroscopy. At pH 4.5, the data are consistent with a low- to high-spin heme transition, with the K99E mutation resulting in a protein with increased peroxidase activity in the absence of or at low concentrations of denaturant. Furthermore, the mobility of the polypeptide chain at pH 4.5 for the wild-type protein has been monitored in the absence and presence of denaturant through heteronuclear NMR experiments. The results are discussed in terms of local stability differences between bacterial and mitochondrial cytochromes c that are inferred from peroxidase activity assays. At pH 7.0, a mixture of misligated heme states arising from protein-based ligands assigned to lysine and histidine is detected. At low denaturant concentrations, these partially unfolded misligated heme forms inhibit the peroxidase activity. Data from the K99E mutation at pH 7.0 indicate that K99 is not involved in heme misligation, whereas histidine coordination is proven by the data from the H118Q variant.
Worrall
Jonathan A R
JA
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
Diederix
Rutger E M
RE
Prudêncio
Miguel
M
Lowe
Christian E
CE
Ciofi-Baffoni
Simone
S
Ubbink
Marcellus
M
Canters
Gerard W
GW
D019943:Amino Acid Substitution; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D019791:Guanidine / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010230:Paracoccus / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D009195:Peroxidase / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D003574:Cytochrome c Group; D006418:Heme; C031732:cytochrome C-550; D009195:Peroxidase; D019791:Guanidine