Rapid mixing apparatus for detection of Fe(I) intermediate(s) of hemoproteins with emphasis on P-450

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Heme catabolism in an important biological process, which is caralyzed by the enzyme heme oxygenase. P-450 is involved in the hydroxylation of steroids, alkanes and in the metabolism of various drugs. <alpha>-Oxprotoheme, in early intermediate in heme catabolism, was synthesized and its autoxidation to biliverdin IX<alpha> was studied using rapid mixing sampling apparatus. The apparatus was prepared for mixing two different samples anaerobically or aerobically below -10゜C, followed by recording electronic spectra and ESR spectra. The experiment demonstrate severel lives of evidence for a unique electronic structure of <alpha>-oxyprotoheme, which is a resonance hybrid between Fe(II)-oxyprotoporphyrin <pi>-neutral radical and Fe(III)-oxyprotorphyrin anion. The data for Fe(III)-oxyprotoporphyrin in basic solvent are consistent with the fromulation of Fe(II)-oxyprotorphyrin <pi>-neutral radical, in which the hydroxy proton is deprotonated to afford theenolate from, therby electron transfer occurring from the enolate anion to the ferric iron to form the Fe(II)-oxyprotoporphyrin <pi>-neutral radical. Such resonance structures depends on the ligand basicity. It is interesting to note that the ESR spectrum of Fe(II)-oxyprotoporphyrin <pi>-neutral radical in basic solvent such as 4-acetylpyridine is quite similar to that for Fe(I)-protoporphyrin which lead us to propose in our prevoius report that cooling to 77K causes one more electron transfer from the porphyrin to Fe(II) to form at Fe(I) compound. In P-450 model system (protoheme-n-butylmercaptide), however, ESR spectrum was silent and showed no Fe(i) spectra which demonstrated one electron transfer from S^- to Fe(II). Instead high oxidation state of Fe(IV) was demonstrated.Using a rapid mixing sampling apparatus, the novel 893 nm intermediate was detected with halflife time of 300 ms during the conversion of <alpha>-oxyprotoheme to <alpha>-versohemochrome.