CHARACTERIZATION OF RECOMBINANT HORSERADISH-PEROXIDASE-C AND 3 SITE-DIRECTED MUTANTS, F41V, F41W, AND R38K, BY RESONANCE RAMAN-SPECTROSCOPY

Resonance Raman spectra are reported for recombinant horseradish peroxidase C (HRP-C*) and three protein variants prepared by in vitro refolding after Escherichia coli expression. The spectra of their Fe-II and Fe-III forms and of their complexes with benzohydroxamic acid (BHA) were recorded at neutral pH. The residues mutated were on the distal [Phe41-->Trp or Val (F41W, F41V) and Arg38-->Lys (R38K)] side of the heme. The spectra give information on the spin and ligation states via the frequencies of the core size marker bands. No detectable modification in the enzyme structure or in the heme group has been observed in the wild-type recombinant HRP-C*. The Fe-III forms of both the recombinant and the plant proteins show the coexistence of a 5-(5-cHS) and a 6-coordinate high-spin (6-cHS) heme, characterized by the anomalous frequencies of certain bands, namely, nu(3) and nu(10), which we attribute to a different degree of distortion of the heme planarity with respect to other heme proteins and model compounds, resulting from external forces such as steric contacts within the protein. This effect is partially relieved upon complexation with BHA or as a result of mutation. F41W and F41V are characterized by an increase in a 6-cHS form at the expense of the 5-cHS species, and the R38K by an increase in both the 6-c high-(HS) and low-spin (LS) hemes. The 6-cHS and -LS species are characterized by normal core size marker band frequencies. The Fe-II-His RR band is at 243 cm(-1) in HRP-C*, the high frequency value being due to hydrogen-bonding interactions between the proximal His170 N-delta and the carboxylate acceptor group on Asp247. Mutation at position 38 causes a downshift of 3 cm(-1) in the nu(Fe-Im) stretching mode, suggesting a weakening of the Fe-Im bond strength. By comparing the results obtained with HRP-C* mutants with those previously reported for the corresponding cytochrome c peroxidase (CCP) mutants, it appears that the distal heme pocket architecture is significantly different in the two peroxidases, although the hydrogen-bonding network coupling the distal and the proximal sides of the heme appears to be conserved. Mutations on the distal side dramatically affect the capability of the protein to bind BHA. F41W and R38K mutants do not bind the substrate, whereas the F41V variant shows a 2-fold increase in affinity. The data are consistent with the current model of the BHA binding site lying toward the distal rather than the proximal side of the heme.