Effect of the His175-->Glu mutation on the heme pocket architecture of cytochrome c peroxidase.
Resonance Raman (RR) and electronic absorption spectra of the ferric and ferrous forms of the His175Glu mutant of cytochrome c peroxidase are reported. At 296 K, the FeIII form is five-coordinate high spin and the resonance Reman spectra are very similar to those obtained for the wild type enzyme, even though in the mutant the Fe atom is bound to an oxygen atom of the Glu residue. The only difference is that the bands due to the out-of-plane modes are very weak, indicating a less distorted heme plane compared to CCP. The absorption spectrum is similar to that of CCP, as far as the Soret and alpha, beta bands are concerned, but the charge-transfer band due to the a2u(pi)-->eg(d pi) transition is 8 nm blue-shifted relative to that of the wild type enzyme, indicating that a more negative ligand is bound to the heme iron. As the temperature is lowered, the five-coordinate heme converts to a six-coordinate high-spin form. The conversion is readily reversible. A temperature effect on the protein structure is proposed that permits the Fe atom to approach the heme plane and to bind the distal water molecule. The results are discussed in terms of the X-ray structure, which shows a different disposition of the distal water molecules in the Glu175 mutant. The RR spectra also show that the heme is more contracted and distorted at 19 K than at room temperature.(ABSTRACT TRUNCATED AT 250 WORDS)
Smulevich
G
G
Dipartimento di Chimica, Universita di Firenze, Italy.
Neri
F
F
Willemsen
O
O
Choudhury
K
K
Marzocchi
M P
MP
Poulos
T L
TL
D000595:Amino Acid Sequence; D001665:Binding Sites; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000378:metabolism*; D018698:Glutamic Acid; D006418:Heme / Q000378:metabolism*; D006639:Histidine; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D014961:X-Ray Diffraction
D011994:Recombinant Proteins; D018698:Glutamic Acid; D006418:Heme; D006639:Histidine; D003578:Cytochrome-c Peroxidase