Alternative carbon monoxide binding modes for horseradish peroxidase studied by resonance Raman spectroscopy

Abstract

Resonance Raman (RR) spectroscopy and infrared spectroscopy have been used to characterize the three vibrational modes, CO and FeC stretching and FeCO bending, for carbon monoxide bound to reduced horseradish peroxidase, with the aid of 13CO and C18O isotope shifts. At high pH, one species, I, is observed, with vFeC = 490 cm-1 and vCO = 1932 cm-1. The absence of a band attributable to .delta.FeCO suggests a linear FeCO unit normal to the heme plane. The data were consistent with I having a strongly H-bonded proximal histidine, as shown by a comparison with imidazole and imidazolate adducts of FeIIPPDME(CO) (PPDME = protoporphyrin IX dimethyl ester), with vFeC = 497 and 492 cm-1 and vCO = 1960 and 1942 cm-1. At low pH an additional species, II, is observed, with vFeC = 537 cm-1, vCO = 1904 cm-1, and .delta.FeCO = 587 cm-1; it is attributed to FeCO that is H bonded to a protonated distal histidine, the H bond strongly lowering vCO and raising vFeC. The appearance of .delta.FeCO in the RR spectrum suggests that the FeCO unit in II is tilted with respect to the heme plane. At low pH, the population of I and II depends on the CO concentration. I dominates at low CO/protein levels but is replaced by II as the amount of CO is increased. This behavior is suggested to arise from secondary binding of CO, which induces a conformation change involving the distal residues of the heme pocket.