Reactions of the oxygen molecule with organic compounds are energetically favorable and constitute some of the most important energy-yielding metabolic processes in biology. The reactions are kinetically stable, however, due to the fact that the ground state of dioxygen is a triplet state and the reactions are therefore spin forbidden. This kinetic stability has prevented the abundant oxygen on earth from reacting uncontrollably with organic materials and has made life on earth possible. Hence, to understand how nature overcomes this kinetic barrier and controls the dioxygen reactivities is of fundamental and vital importance. In biology, activation of dioxygen is generally achieved by complexing the oxygen molecule with metal cofactors in proteins. Up until now, the best known and most studied protein systems that catalyze O2-dependent reactions are probably the heme-containing proteins. More recently, however, it has become obvious that proteins containing carboxylate-bridged diiron centers represent yet another group of proteins that possesses catalytic capabilities as diverse as those of hemoproteins. For the past few years, we have applied optical, Mossbauer and EPR spectroscopies to characterize the carboxylate-bridged diiron centers in the wild-type and recombinant rubrerythrin. We have also used the rapid freeze-quench Mussbauer and EPR methods to study (a) the reconstitution of the diiron-tyrosyl radical cofactor in the R2 subunit of the ribonucleotide reductase (RNR) from Escherichia coli, and (b) the reaction of O2 with the diferrous center in methane monooxygenase (MMO) from Methylococus capsulatus (Bath). Several novel, kinetically competent, transient intermediates have been identified and partially characterized. In this application, we request support to continue our kinetic and spectroscopic investigations on rubrerythrin, RNR, and MMO, and to extend our research program to include mechanistic studies on the ferroxidase activity of ferritin. Recent studies in ferritin suggest that a carboxylate-bridged diiron center may be involved in the Fe(II) oxidation during the initial stage of the ferrihydrite core formation. Our specific objective is to obtain mechanistic information on the reaction of the ferrous centers with O2 in these protein systems. Our general goal is to understand how biology uses protein structure to control the reactivity of complex metal centers, in this case, controlling the carboxylate-bridged diiron center to activate O2 for a variety of catalytic functions.
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