The R2 subunit of Escherichia coli ribonucleotide reductase (RNR) contains a stable tyrosyl radical (•Y122) diferric cluster cofactor. Earlier studies on the cofactor assembly reaction detected a paramagnetic intermediate, X, that was found to be kinetically competent to oxidize Y122. Studies using rapid freeze-quench (RFQ) Mössbauer and EPR spectroscopies led to the proposal that X is comprised of two high spin ferric ions and a S = 1/2 ligand radical, mutually spin coupled to give a S = 1/2 ground state (Ravi, N.; Bollinger, J. M., Jr.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J. J. Am. Chem. Soc. 1994, 116, 8007−8014). An extension of RFQ methodology to Q-band ENDOR spectroscopy using 57Fe has shown that one of the irons has a very nearly isotropic hyperfine tensor (A(FeA) = −[74.2(2), 72.2(2), 73.2(2)] MHz) as expected for FeIII, but that the other iron site displays considerable anisotropy (A(FeB) = +[27.5(2), 36.8(2), 36.8(2)] MHz), indicative of substantial FeIV character. Reanalysis of the Mössbauer data using these results leads to isomer shifts of δ(FeA) = 0.56(3) mm/s and δ(FeB) = 0.26(4) mm/s. Based on the hyperfine anisotropy of FeB plus the reduced isomer shift, X is now best described as a spin-coupled FeIII/FeIV center without a radical, but with significant spin delocalization onto the oxygen ligand(s).

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Reactive halogen species (RHS; X 2 and HOX, where X represents Cl, Br, or I) are metabolites mediated by neutrophil activation and its accompanying respiratory burst. We have investigated the interaction between RHS and mitochondrial cytochrome c (cyt c) by using electrospray mass spectrometry and electron spin resonance (ESR). When the purified cyt c was reacted with an excess amount of hypochlorous acid (HOCl) at pH 7.4, the peroxidase activity of cyt c was increased by 4.5-, 6.9-, and 8.6-fold at molar ratios (HOCl/cyt c) of 2, 4, and 8, respectively. In comparison with native cyt c, the mass spectra obtained from the HOCl-treated cyt c revealed that oxygen is covalently incorporated into Respiratory burst during phagocytosis of bacteria by neutrophils is a physiological response (1). It is well known that during the respiratory burst, a number of enzymatic processes are activated, which then act to produce oxygen metabolites. For example, the hexose monophosphate shunt is activated to increase glucose oxidation and production of CO 2 and NADPH (1). Superoxide anion is generated by activation of NADPH oxidase; superoxide then dismutates to hydrogen peroxide (1).However, the respiratory burst of neutrophils or monocytes can also mediate the metabolism of halide and generate reactive halogen species (RHS) 1 such as HOX and X 2 (where X represents Cl, Br, or I) (1, 2). In the presence of H 2 O 2 , a halide anion is a substrate in the peroxidative reaction catalyzed by myeloperoxidase (MPO) released from the primary granule (1, 3, 4). The halide that is commonly most abundant is Cl Ϫ (physiological concentration of 100 mM), and the reactive MPO system is unique in oxidizing chloride ion (Reactions 1 and 2). MPO(Fe III