Conformational change and histidine control of heme chemistry in cytochrome c peroxidase: resonance Raman evidence from Leu-52 and Gly-181 mutants of cytochrome c peroxidase.
Resonance Raman (RR) spectra are reported for Fe(III), Fe(II), and Fe(II)CO forms of site-directed mutants of the cytochrome c peroxidase variant CCP(MI), cloned in Escherichia coli. The Fe(II) form is five-coordinate (5-c) and high-spin at low pH, but it is six-coordinate (6-c) and low-spin at high pH except when the distal His-52 residue is replaced with Leu, showing the sixth ligand to be the His-52 imidazole. Although the Leu-52 mutant stays 5-c, it does undergo an alkaline transition, as revealed by upshifts and broadening of bands assigned to vinyl C = C stretching (1620 cm-1) and C beta-vinyl bending (402 cm-1). Similar changes are seen for CCP(MI) and other mutants. Thus the alkaline transition induces a conformational change that affects the vinyl groups, probably through changes in their orientation, and that permits the His-52 imidazole to bind the Fe. The RR band arising from the stretching of the proximal Fe(II)-imidazole bond contains components at ca. 235 and 245 cm-1 for CCP(MI), which are believed to reflect a double well potential for the H-bond between the proximal His-175 imidazole and the Asp-235 carboxylate group. Loss of this H-bond by mutation of Asp-235 to Asn results in the loss of these two bands and their replacement by a single band at 205 cm-1. Although the Fe(II)-imidazole stretching mode cannot be observed in the 6-c alkaline form of the enzyme, the sixth ligand in the alkaline form of CCP(MI) is photolabile, and the status of the Fe(II)-imidazole bond can be determined in the resulting 5-c-photoproduct. For CCP(MI) at alkaline pH, the conformation change induces an increase in the 235/245-cm-1 ratio, reflecting a perturbation of the H-bond potential. In the His-52----Leu mutant, a 205-cm-1 band appears along with the 235/245-cm-1 doublet at alkaline pH, indicating partial loss of the proximal H-bond due to the distal alteration. The effect of mutations that perturb the H-bonding network that extends from the distal to the proximal side of the heme is more dramatic: at alkaline pH, His-181----Gly, Arg-48----Leu, and Trp-51----Phe mutants show an Fe(II)-imidazole stretching mode at 205 cm-1 exclusively, indicating complete loss of the proximal Asp-235-His-175 H-bond.(ABSTRACT TRUNCATED AT 400 WORDS)
Smulevich
G
G
Dipartimento di Chimica, Università di Firenze, Italy.
Miller
M A
MA
Kraut
J
J
Spiro
T G
TG
D000475:Alkenes / Q000737:chemistry; D001483:Base Sequence; D002248:Carbon Monoxide / Q000737:chemistry; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D004252:DNA Mutational Analysis; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000737:chemistry; D008024:Ligands; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013055:Spectrophotometry, Infrared; D013059:Spectrum Analysis, Raman; D013329:Structure-Activity Relationship; D014867:Water / Q000737:chemistry
D000475:Alkenes; D005290:Ferric Compounds; D005296:Ferrous Compounds; D008024:Ligands; D009841:Oligonucleotides; D011994:Recombinant Proteins; D014867:Water; D006418:Heme; D006639:Histidine; D002248:Carbon Monoxide; D003578:Cytochrome-c Peroxidase; D007930:Leucine; D005998:Glycine