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 co mplexes of HRPC His 42 --> Leu and Arg 38 --> Leu mutants were charact erized at pH values ranging from 3.6 to 9.5. The vibrational frequenci es of the Fe-C stretching and Fe-C-O bending modes have been identifie d by isotopic substitution. Both His 42 --> Leu and Arg 38 --> Leu add ucts with CO displayed a single Fe-C stretching band, whereas both rec ombinant and wild-type HRPC-CO have two bands, corresponding to differ ent conformers.` This comparison suggests that CO is H-bonded either t o the distal Arg or to the distal His in the two conformers. An acid t ransition, common to the wild-type protein, was observed for both muta nts. This indicates that these distal amino acids do not influence the acid transition. On the contrary, an alkaline transition was only obs erved for the Arg 38 --> Leu mutant, which suggests that distal His is involved in the alkaline transition of HRPC-CO complex. The spectrosc opic information is found to be consistent with the X-ray structure of ferric HRPC. A comparison with the CO complexes of cytochrome c perox idase and myoglobin is performed, which displays the functional signif icance of the structural differences between peroxidase classes I and III and between peroxidases and globins, respectively.