EPR and ENDOR studies of cryoreduced compounds II of Peroxidases and myoglobin. Proton-coupled electron transfer and protonation status of ferryl

The nature of the [Fe(IV)-O] center in hemoprotein Compounds II has recently received considerable attention, as several experimental and theoretical investigations have suggested that this group is not necessarily the traditionally assumed ferryl ion, -[Fe(IV)=O](2+), but can be the protonated ferryl, [Fe(IV)-OH](3+). We show here that cryoreduction of the EPR-silent Compound 11 by gamma-irradiation at 77 K produces Fe(III) species retaining the structure of the precursor [Fe(IV)=O](2+) or [Fe(IV)-OH]31, and that the properties of the cryogenerated species provide a report on structural features and the protonation state of the parent Compound II when studied by EPR and H-1 and N-14 ENDOR spectroscopies. To give the broadest view of the properties of Compounds H we have carried out such measurements on cryoreduced Compounds II of HRP, Mb, DHP and CPO and on CCP Compound ES. EPR and ENDOR spectra of cryoreduced HRP 11, CPO 11 and CCP ES are characteristic of low-spin hydroxy-Fe(III) heme species. In contrast, cryoreduced "globins", Mb II, Hb II, and DHP II, show EPR spectra having lower rhombicity. In addition the cryogenerated ferric "globin" species display strongly coupled exchangeable 1H ENDOR signals, with A(max) similar to 20 MHz and a(iso) similar to 14 MHz, both substantially greater than for hydroxide/water ligand protons. Upon annealing at T > 180 K the cryoreduced globin compounds II relax to the low-spin hydroxy-ferric form with a solvent kinetic isotope effect, KIE > 6. The results presented here together with published resonance Raman and Mossbauer data suggest that the high-valent iron center of globin and HRP compounds II, as well as of CCP ES, is [Fe(IV)=O](2+), and that its cryoreduction produces [Fe(III)-O](+). Instead, as proposed by Green and co-workers - (1), CPO II contains [Fe(IV)-OH](3+) which forms [Fe(III)-OH](2+) upon radiolysis. The [Fe(III)-O](+) generated by cryoreduction of HRP 11 and CCP ES protonate at 77 K, presumably because the heme is linked to a distal-pocket hydrogen bonding/protondelivery network through an H-bond to the "oxide" ligand. The data also indicate that Mb and HRP compounds II exist as two major conformational substates.