RESONANCE RAMAN AND EPR SPECTROSCOPIC STUDIES ON HEME HEME OXYGENASE COMPLEXES

The binding of ferrous and ferric hemes and manganese(II)- and mangane se(III)-substituted hemes to heme oxygenase has been investigated by o ptical absorption, resonance Raman, and EPR spectroscopy. The results are consistent with the presence of a six-coordinate heme moiety ligat ed to an essential histidine ligand and a water molecule. The latter i onizes with a pK(a) almost-equal-to 8.0 to give a mixture of high-spin and low-spin six-coordinate hydroxo adducts. Addition of excess cyani de converts the heme to a hexacoordinate low-spin species. The resonan ce Raman spectrum of the ferrous heme-heme oxygenase complex and that of the Mn(II)protoporphyrin-heme oxygenase complex shows bands at 216 and 212 cm-1, respectively, that are assigned to the metal-histidine s tretching mode. The EPR spectrum of the oxidized heme-heme oxygenase c omplex has a strongly axial signal with g(parallel-to) almost-equal-to 6 and g(perpendicular-to) almost-equal-to 2. (NO)-N-14 and (NO)-N-15 adducts of ferrous heme-heme oxygenase exhibit EPR hyperfine splitting s of approximately 20 and approximately 25 Gauss, respectively. In add ition, both nitrosyl complexes show additional superhyperfine splittin gs of approximately 7 Gauss from spin-spin interaction with the proxim al histidine nitrogen. The heme environment in the heme-heme oxygenase enzyme-substrate complex has spectroscopic properties similar to thos e of the heme in myoglobin. Hence, there is neither a strongly electro n-donating fifth (proximal) ligand nor an electron-withdrawing network on the distal side of the heme moiety comparable to that for cytochro mes P-450 and peroxidases. This observation has profound implications about the nature of the oxygen-activating process in the heme --> bili verdin reaction that are discussed in this paper.