The distal cavity structure of carbonyl horseradish peroxidase as probed by the resonance Raman spectra of His 42 Leu and Arg 38 Leu mutants.
CO ligation to horseradish peroxidase C (HRPC) was studied by means of site-directed mutagenesis and resonance Raman spectroscopy. The CO complexes of HRPC His 42 --> Leu and Arg 38 --> Leu mutants were characterized at pH values ranging from 3.6 to 9.5. The vibrational frequencies of the Fe-C stretching and Fe-C-O bending modes have been identified by isotopic substitution. Both His 42 --> Leu and Arg 38 --> Leu adducts with CO displayed a single Fe-C stretching band, whereas both recombinant and wild-type HRPC-CO have two bands, corresponding to different conformers. This comparison suggests that CO is H-bonded either to the distal Arg or to the distal His in the two conformers. An acid transition, common to the wild-type protein, was observed for both mutants. This indicates that these distal amino acids do not influence the acid transition. On the contrary, an alkaline transition was only observed for the Arg 38 --> Leu mutant, which suggests that distal His is involved in the alkaline transition of HRPC-CO complex. The spectroscopic information is found to be consistent with the X-ray structure of ferric HRPC. A comparison with the CO complexes of cytochrome c peroxidase and myoglobin is performed, which displays the functional significance of the structural differences between peroxidase classes I and III and between peroxidases and globins, respectively.
Feis
A
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Dipartimento di Chimica, Università di Firenze, Italy.
Rodriguez-Lopez
J N
JN
Thorneley
R N
RN
Smulevich
G
G
D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics*; D002248:Carbon Monoxide / Q000737:chemistry; D006639:Histidine / Q000235:genetics*; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics; D007930:Leucine / Q000235:genetics*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013059:Spectrum Analysis, Raman
D007527:Isoenzymes; D011994:Recombinant Proteins; D006639:Histidine; D002248:Carbon Monoxide; D001120:Arginine; D006735:Horseradish Peroxidase; D007930:Leucine