PROTON NUCLEAR OVERHAUSER EFFECT STUDY OF THE HEME ACTIVE-SITE STRUCTURE OF CHLOROPEROXIDASE

Chloroperoxidase, a glycoprotein from the mold Caldariomyces fumago, has been investigated in its ferric low-spin cyanide-ligated form through use of nuclear Overhauser effect (NOE) spectroscopy to provide information on the heme pocket electronic/molecular structure. Spin-lattice relaxation times for the hyperfine-shifted heme resonances were found to be three times less than those in horseradish peroxidase. This must reflect a slower electronic relaxation rate for chloroperoxidase than for horseradish peroxidase as a consequence of axial ligation of cysteine in the former versus histidine in the latter enzyme. Isoenzymes A1 and A2 of chloroperoxidase show the largest chemical shift differences near the heme propionate on the basis of NOE measurements. This suggests that the primary structure differences for the two isoenzymes are communicated to the heme group through the ring propionate substituents. A downfield peak has been detected in chloroperoxidase with chemical shift, T1, and line width characteristics similar to those of the C(epsilon)-H proton of the distal histidine in horseradish peroxidase. This finding is in agreement with a previous suggestion for a distal histidine residue. The NOE pattern and T1's of the peaks in the 0.0 to -5.0 ppm upfield region are consistent with the presence of an arginine amino acid residue in the heme pocket near either the 1-CH3 or 3-CH3 group. Existence of catalytically important distal histidine and arginine amino acid residues in chloroperoxidase shows it to be structurally similar to peroxidases rather than to the often compared monooxygenase, cytochrome P-450. This result supports the earlier conclusions of Sono et al. [Sono, M., Dawson, J. H., Hall, K., & Hager, L. P. (1986) Biochemistry 25, 347-356].