Heme iron active sites are present in a wide variety of proteins and enzymes. The different biological functions they perform include oxygen binding, storage, transfer, and activation for hydroxylation, peroxidation, and desaturation. This project examines the molecular biochemistry exhibited by two structurally diverse heme-containing enzymes that oxidize either free tryptophan or protein-bound tryptophan residues. Tryptophan 2,3-dioxygenase (TDO) inserts two oxygen atoms into free tryptophan in a four electron oxidizing process by a b-type heme cofactor. This enzyme represents a potentially new hemoprotein dioxygenase superfamily whose oxygenase activity remains poorly understood. MauG is a novel enzyme that utilizes two c-type hemes to catalyze a posttranslational modification of a 119 KDa protein. Such a modification endows endogenous tryptophan residues with a new catalytic activity. The MauG-catalyzed reaction is a six-electron oxidation process and the utilization of two c-type hemes to perform a hydroxylation reaction and the subsequent oxidation reactions are unprecedented. Novel high valent iron intermediates have been trapped from these tryptophan oxidizing enzymes. This research will establish a better definition of the requirements needed to attain the unusual Fe(IV) intermediates as well as elucidate their relation to oxidizing the free or protein-bound tryptophan substrates. A biochemical and spectroscopic approach employing site-directed mutagenesis, electron paramagnetic resonance (EPR) spectroscopy, Mössbauer spectroscopy, mass spectrometric analysis, and structural determination as well as quantum mechanics computational methods will be utilized to study the electronic structure of the intermediates in these enzymes. These studies are directed toward obtaining detailed insight into electronic and geometric structural contributions to the formation and stabilization of high valent iron intermediates, the electronic structure of reactive oxygen intermediates, and the heme-dependent tryptophan oxidizing mechanisms contrasts to non-heme catalysis.
Broader Impacts: This research will provide the opportunity for motivated students, including underrepresented populations, to participate in the frontier of molecular biochemistry research. Students exposure to an array of biochemical and biophysical techniques will be integrated with hands-on training. A video tutorial of electron paramagnetic resonance (EPR) spectroscopy will be developed during the experimental process to provide a thorough demonstration of EPR-centered techniques in studying the chemical properties of important biomolecular systems.
In recent years, it has come to light that many crucial biological processes are often mediated by some of the culprits, activated oxygen species and free radicals, which have been implicated in aging and oxidative stress. The dual roles of these reactive species as the leading cause of non-specific cell damage and as useful catalytic intermediates in biochemical reactions necessitate that their use in biological systems should be highly specific and strictly controlled. In particular, radical generation and long-range electron transfer as a means to transport oxidizing equivalents is increasingly regarded as an effective strategy for biological systems to perform demanding redox reactions in a controlled manner. To pursue an evidence-supported understanding, we have performed experimental and theoretical studies on the mechanism of two heme-dependent enzymes for oxidation of essential amino acid tryptophan under the support of NSF MCB- 0843537 award. In the enzyme that oxidizes protein-bound tryptophan, we have made several pieces of breakthroughs that are high impact to the field. We have discovered how an enzyme oxidizes two buried tryptophan residues in a large protein that is about 4-fold bigger than the enzyme, through a long distance. We have discovered an unprecedented powerful oxidant made by the enzyme, which we termed it bis-Fe(IV). This intermediate is chemically equivalent to an Fe(V) species but it is much longer lived for handling a large substrate rather than a small organic compound. This enzyme-based oxidant stabilizes itself through a so-called chemical charge resonance phenomenon which has not previously seen in biology systems. This oxidant orchestrates the actual oxidation process through a long distance electron transfers and produce a tryptophan-based di-radical intermediate on the large substrate to complete the long-range remote catalysis. We have captured and characterized all the key intermediates during the funding period of this NSF award. In the enzyme that oxidizes free (meaning not part of a protein) tryptophan, we have established a catalytic mechanism by which the enzyme orchestrates the two substrates in a manner that molecular oxygen bridges the metal ion and tryptophan, leading to a weaker oxygen-oxygen bond. The cleavage of the oxygen-oxygen bond lead to oxygen insertion of one oxygen and a highly reactive iron-bound oxygen intermediate, namely, Fe(IV)=oxo, which subsequently insert the oxygen to the tryptophan again. This enzyme avoids producing single electron-based free radicals. During the stud of this latter enzyme, we have also solved an over sixty years long-standing problem. The oxygen activation enzyme is active in the reduced state with the iron ion at the +2 valence state; once oxidized to +3 it is inactive. However, the inactive enzyme was found to become active once it is further oxidized in the presence of tryptophan by an oxidant such as hydrogen peroxide. We discovered an enzyme reactivation mechanism which may be valid for many of the iron-dependent, oxygen activation enzymes. Overall, we have conducted a highly productive research that leads to 24 peer-reviewed journal publications, 5 dissertations, and significant contributions to the human resources and recruiting underrepresented populations to science, curriculum development, and also measurable contributions to the instuitional infrastructure and information systems.
