Research in the Pierce group employs modern biophysical techniques to investigate inorganic chemistry relevant to biology. In particular, dual-mode EPR spectroscopy, in combination with spectroscopic simulations, is a highly sensitive method for probing the active site electronic structure of metalloenzymes. Specific topics of interest to our group include the oxidation of sulfur-bearing bimolecules, pathogenic adaptation to oxidative/nitrosative stress, and non-heme enzymes involved in tRNA modification.

Cysteine dioxygenase (CDO) and cysteamine (2-aminoethanethiol) dioxygenase (ADO) are the only known mammalian thiol dioxoygenase (TDO) enzymes.  TDO enzymes use a single Fe(II) ion within their active site to catalyze the oxygen-dependent oxidation of sulfur-bearing amino acid derivatives without the need for an external electron source.  Of the TDO enzymes, mammalian CDO is the best characterized.  Until recently, the catabolic dissimilation of L-cysteine to produce inorganic sulfate, pyruvate, hypotaurine, and taurine was believed to be unique within the domain of eukaryotics.  However, a number of bacterial TDO enzymes have now been identified, suggesting that the ability to oxidize excess thiols is advantageous for survival.  Indeed, the biochemistry associated with sulfur metabolism has increasingly become relevant for the development of pathogenic drug targets as well as therapies for cancer and inflammatory disease.  In mammals, imbalances in sulfur metabolism are frequently used as a biomarker for the identification of neurological and autoimmune disorders, such as Autism, and Parkinson’s, Alzheimer’s disease. 

Currently, the Pierce group is working toward the development of a mechanistic model for sulfur-oxidation catalyzed by TDO enzymes.  An integrated biophysical/inorganic approach is applied to the study of these enzymes, relying on techniques such as steady-state and pre-steady state kinetics, site-directed mutagenesis, mass spectrometry, UV-visible, and EPR spectroscopy.

The ability to resist the nitric oxide (NO) synthesized by host phagocytes is a virulence determinant in some pathogens.  In particular, iron-sulfur proteins are particular susceptible to oxidative and nitrosative induced inactivation.  Exposure to NO induces up-regulation of genes required for NO detoxification and repair of NO-mediated damage.  One such gene in E. coli (ytfE) is phylogenetically widely distributed and is consistently up-regulated by NO in different species.  (This gene has also been referred to as norA).  The protein expressed by ytfE is diiron hemerythrin-like protein that is transcription regulated by the NO-sensitive repressor, NsrR.  It has been proposed that the ytfE metalloprotein is plays a direct role the repair of iron-sulfur enzymes damaged by NO.  To date, the mechanism of Fe-S repair mediated by ytfE remains unclear.  To shed light on the enigmatic but important function of ytfE, the Pierce group employs UV-visible and dual-mode X-band EPR spectroscopy to simultaneously observe changes in the diiron cluster of yftE and the integrity of NO-damaged Fe-S clusters.

Chemical modifications of nucleosides within transfer RNAs (tRNAs) occur ubiquitously throughout nature. Most frequently modified are the regions near the tRNA wobble-position and adjacent to the anticodon position. Presumably, these covalent modifications increase the specificity for the modified tRNA by altering the conformation of the tRNA molecule near the modified position. A variety of enzymes have been identified which carry out post-transcriptional modifications on tRNA, all of which demonstrate remarkable specificity in order to modify a single nucleoside on the tRNA substrate. One particularly remarkable modification of tRNA involves the oxygen-dependent hydroxylation of a (2-methythio-N-6-isopentyl adenosine) found at position 37. The enzyme which catalyzes this reaction is the product of the miaE gene in Salmonella typhimurium. Spectroscopic characterization of recombinantly expressed MiaE identified that this enzyme contains a non-heme diiron active site analogous to members of the class I family of non-heme diiron enzymes. Other members of this family include the bacterial multicomponent monooxygenases [methane monooxygenase (MMO)] and the soluble stearoyl-ACP D-9-desaturase (D9D). The Pierce group is working toward investigating the role of protein-RNA factors which may modulate the reactivity of the diiron cluster and give rise to the remarkable substrate specificity.