Project summary Iron enzymes play major roles in O2 activation in biology. These divide into four classes based on their active site structures that reflect their mode of O2 activation: the non-cofactor dependent mononuclear non-heme iron (MNHFe) enzymes, the cofactor (α-ketoglutarate (α-KG) and pterin) dependent MNHFe enzymes, the binuclear NHFe enzymes and the O2/H2O2 activating heme enzymes. Crystal structures and oxygen reaction intermediates exist for metalloenzymes in all four classes. Over the years, we have developed new spectroscopic methods enabling the detailed study of the NHFeII active sites, and the geometric and electronic structures of their O2 intermediates and have now developed a method to quantitatively study the iron center in the highly covalent and chromophoric heme environment. Among our accomplishments in the past 5 years are: 1) determined that FeIII-O2- species are the reactive intermediates in all the subclasses of non-cofactor dependent MNHFe enzymes; 2) defined the O2 reaction coordinates to generate the FeIV=O intermediates in both the α-KG and pterin dependent enzymes; 3) for the α-KG dependent subclass, defined the geometric and electronic structures of their FeIV=O enzyme intermediates and how these direct halogenation over the thermodynamically favored hydroxylation in the halogenases; 4) showed that in contrast to the MNHFe enzymes, hydroperoxide intermediates are active in the binuclear NHFe enzymes for direct reaction with substrates; 5) for methane monooxygenase, where the peroxo-biferric intermediate rapidly converts to a high-valent 2FeIV-oxo intermediate Q, we have determined the structure of Q (a topic of current debate) and provided insight into its high reactivity with methane; 6) used the spectroscopic method we have now developed for iron in heme environments to determine experimentally the computationally controversial electronic structure of oxyhemoglobin; 7) and extended this method to analyze active sites with strong Fe-oxo bonds. Our studies are now directed toward completing the reaction coordinates of the four classes and their subclasses, understanding O2 activation at the superoxo, peroxo and FeIV-oxo levels, determining the role of the second iron in the enhanced reactivity of the binuclear NHFe enzymes, and understanding the differences in the activation and selectivity of high-valent iron-oxo intermediates in mononuclear NH, binuclear NH and heme enzyme active sites.