Oxygenated intermediate and carbonyl species of cytochrome o (Vitreoscilla). Characterization by infrared spectroscopy.

Oxygenated and carbonyl species of cytochrome o (Vitreoscilla) have been studied by infrared spectroscopy to elucidate the nature of Fe02 and FeCO bonding.Under turnover conditions in which NADH, 0 2 , and NADH-cytochrome o reductase were present at 4 "C, a characteristic "oxy" visible spectrum ( X , , , 415, 543,and 577 nm) is observed and an infrared band due to bound 1 6 0 2 is found at 1134 cm" with a half-band width (AvllZ) of 17 cm" and an integrated intensity (B) of -10 m"' cm-2.When the available oxygen becomes exhausted, the infrared band disappears and the characteristic "deoxy" visible spectrum ( X , , ,423 and 553 nm) appears.With *'02, the infrared band is shifted to 1078 cm".Only a single infrared band is seen for either '*02 or "02 in contrast to oxyhemoglobins and oxymyglobins for which multiple bands are found.These infrared spectra provide the first direct evidence for the nature of 0 2 bonding to an oxidase.Since the frequencies for 0 2 vibrations are in similar regions, bent-end-on oxygenyl-type bonding between ferrous iron and dioxygen must occur in oxycytochrome o as well as in oxyhemoglobin and oxymyoglobins.However, there are characteristic differences in infrared spectra which indicate significant bonding differences between the three hemeproteins.CO bound to the fully reduced enzyme at 26 "C exhibits a stretch band with vc0 = 1964 cm-', A V ~, ~ = 9 cm", and B = 14 m"' crn-'.The intensity of the CO band is unusually sensitive to changes in temperature; B increased to 21 m"' cm-2 as the sample temperature was lowered from 26 to 4 "C.This C-0 stretch frequency is much higher than the major bands of hemoglobinAC0 (1951 cm") or bovine myoglobinCO (1944 em") whereas this vCo is nearly identical to that found for the carbonyl of another oxidase, cytochrome c oxidase.The band width is much less for cytochrome c oxidase (Av1,2 = 4 cm") which is consistent with a more stable and less exposed ligand-binding site.These highly characteristic infrared parameters for bound O2 and CO indicate unique features for ligand binding to cytochrome o compared with the other hemeproteins that have been similarly examined by infrared spectroscopy.