Direct infrared detection of the covalently ring linked his-tyr structure in the active site of the heme-copper oxidases

Infrared spectroscopy, isotopic labeling ([¹⁵N_δε]histidine and ring-deuterated tyrosine), synthetic model studies, and normal mode calculations are employed to search for the spectroscopic signatures of the unique, covalently linked (His Nε-Cε Tyr) biting structure in the heme-copper oxidases. The specific enzyme examined is the cytochrome b0₃ quinol oxidase of E. coli. Infrared features of histidine and tyrosine are identified in the frequency regions of imidazole and phenol ring stretching modes (1350-1650 cm^-1) and C-H and N-H stretching modes as well. as overtones and combinations (> 3000 cm^-1). Two of these, at ca. 1480 and 1550 cm^-1, and their combination tones between 3010 and 3040 cm^-1, are definitively identified with the biting structure involving H284 and Y288 in the E. coli enzyme. Studies of a synthetic analogue of the H-Y structure, 4-methylimidazole covalently linked to p-cresol, show that a feature near 1540 cm^-1 is unique to the biring structure and is absent from the infrared spectrum of 4-methylimidazole or p-cresol alone. This feature is readily detectable by infrared difference techniques, and offers a direct spectroscopic probe for potential radical production involving the H-Y structure in the O₂ reduction cycle of the oxidases.