Benzohydroxamic Acid-Peroxidase Complexes: Spectroscopic Characterization of a Novel Heme Spin Species

A distinctive characteristic of the class III heme peroxidases of the plant peroxidase superfamily is the presence of a pentacoordinate quantum mechanically mixed-spin heme state resulting from the admixture of S = 5/2 and S = 3/2 states. This is not observed in class I or II peroxidases and, in fact, is a very rare heme spin state. The corresponding hexacoordinate quantum mechanically mixed-spin state is even more uncommon and has not been observed in heme proteins. The presence of the pentacoordinate form in the class III peroxidases suggested that they could be ideal candidates to display also the six-coordinate quantum mechanically mixed-spin state. With this possibility in mind, the benzohydroxamic acid complexes of the class III peroxidases horseradish isoenzyme C and A2 and soybean peroxidase are studied by electronic absorption, resonance Raman, and EPR spectroscopy at room and low temperatures. The results are compared with those obtained for Coprinus cinereus peroxidase which belongs to class II. The binding of benzohydroxamic acid to horseradish peroxidase A2 and soybean peroxidase is found to be 400- and 500- fold weaker, respectively, than that to horseradish peroxidase C. The data show that the binding of benzohydroxamic acid induces the formation of a six-coordinate heme and that the complex is not appreciably altered by lowering the temperature. The C. cinereus peroxidase-benzohydroxamic acid complex shows resonance Raman and EPR spectra which are readily identified as being due to six-coordinate high-spin heme, whereas the benzohydroxamic acid complexes with the other peroxidases are characterized by Raman core size marker bands at higher frequencies than expected for six-coordinate high spin. The magnitude of the increase in frequency of the resonance Raman bands follows the order horseradish peroxidase C, soybean peroxidase, to horseradish peroxidase A2, and, concomitantly, the g perpendicular to EPR value decreases in the same order. It is proposed that these spectroscopic features are characteristic of a six-coordinate form in a quantum mechanically mixed-spin state, with an increasing contribution from an S = 3/2 State in the same order as the increasing Raman frequencies.