Education

B.S., 1995, College of William and Mary
Ph.D., 2001, Northwestern University

Honors and Awards

Research Corporation Cottrell Scholar, 2008-2010

Research Interests

Research Areas: Microbial metal metabolism, bioinorganic chemistry, microbial physiology, and microbial genetics; biochemical mechanisms of Fe-S cluster assembly; characterization of transition metal acquisition, trafficking, and storage systems and their transcriptional and post-transcriptional regulation during environmental stress.

Metal Trafficking and Metal Cofactor Assembly Under Stress Conditions: My broad research goal is to understand how homeostasis of essential transition metals is maintained in response to environmental stresses. Due to their unique chemical properties, transition metals such as copper, iron, and zinc are critical cofactors in the active sites of enzymes and as structural components in proteins. However, many of these essential metals are toxic when present in excess, indicating a requirement for the cell to maintain a fairly narrow intracellular concentration of each metal. In addition, metal metabolism may be altered by environmental stress through multiple mechanisms. Cells can adjust transport and storage of the metal in response to the stress, either through increased uptake, efflux, or expression of metal storage proteins. The metal or metal cofactor may be directly modified, for instance by oxidation or reduction, leading to a subsequent change in reactivity, ligand affinity, or bioavailability. Conversely, the proteins or other biomolecules that interact with the metal or metal cofactor may themselves be altered by the stress, causing a change in metal metabolism such as release of metal from an active site. Defining the biochemical strategies used by organisms to maintain metal homeostasis under stress will provide insight into critical areas ranging from bacterial pathogenesis to human disease.

The suf pathway and Fe-S cluster assembly under stress: Fe-S clusters, which contain inorganic sulfur and iron, play key roles in electron transport, as active site cofactors in TCA cycle enzymes, and as exquisite sensors of oxygen and oxygen radicals in stress-responsive transcription factors. However, Fe-S clusters are perturbed by multiple stress conditions. During oxidative stress, superoxide anion (O^2•-) can damage 4Fe-4S clusters leading to cluster degradation and release of iron. Therefore, Fe-S clusters are assembled in vivo via intricate biosynthetic pathways. The Fe-S cluster assembly pathway encoded by the sufABCDSE operon is required to assemble Fe-S clusters during iron starvation or oxidative stress, conditions known to disrupt Fe-S clusters in vivo. To determine the biochemical mechanisms used by the suf pathway to achieve this feat, we have purified all six of the suf-encoded proteins. We have found that SufB, SufC and SufD, co-purify as a stable complex. This three-protein complex interacts with the SufE protein to dramatically enhance sulfur donation by the SufS cysteine desulfurase enzyme. SufE acts as a sulfur transfer partner and together with the SufBCD complex, which comprises a novel sulfur transfer pathway for Fe-S cluster assembly under stress conditions. Further genetic, regulatory, and biochemical analysis will elucidate how the suf gene products are adapted to acquire iron and sulfur for construction of Fe-S clusters during iron starvation and oxidative stress.

Metal-responsive Gene Regulation: Transition metal homeostasis is a key process for all forms of life. Metal homeostasis can be disrupted by a variety of environmental or genetic factors. For example, oxygen can alter the oxidation state of some transition metals, such as iron and copper, thereby altering their bioavailability and toxicity. In addition, the requirement for multiple transition metals for correct cell function can be problematic if some transition metals compete with each other for binding to similar protein active sites. Iron is critical for growth due to the need for iron in cofactors like heme and iron-sulfur (Fe-S) clusters. However, iron has limited bioavailability in the environment and iron homeostasis is disrupted by oxidative stress. Iron can also be disrupted by excess levels of other metals, such as cobalt and copper, that compete with iron for incorporation into Fe-S clusters. Under anaerobic conditions in bacteria, iron is combined with nickel in the active sites of the Ni-Fe hydrogenase family of enzymes. During anaerobic growth bacteria increase nickel uptake to provide it for hydrogenase maturation. We have found that excess nickel is toxic to E. coli in part due to disruption of iron homeostasis. We have also identified a transcription factor YqjI that regulates iron homeostasis genes in response to cellular nickel accumulation under anaerobic conditions. YqjI is a nickel-dependent metalloregulatory protein that coordinates control of the ferric reductase YqjH in order to balance iron and nickel uptake under anaerobic conditions. We are characterizing the biochemical mechanism for nickel regulation of YqjI, the transcriptional control of yqjH expression, and the in vivo connections between iron and nickel metabolism under anaerobic conditions.

Selected Publications

Kim, D.; Singh, H.; Dai, Y.; Dong, G.; Busenlehner, L.S.; Outten, F.W.; Frantom, P.A.  Changes in protein dynamics in Escherichia coli SufS reveal a possible conserved regulatory mechanism in Type II Cysteine Desulfurase systems.  Biochemistry. 2018, In press.  DOI: 10.1021/acs.biochem.7b01275.

Blahut, M.; Dzul, S.; Wang S, Kandegedara, A.; Grossoehme, N.E.; Stemmler, T.; Outten, F.W.  Conserved cysteine residues are necessary for nickel-induced allosteric regulation of the metalloregulatory protein YqjI (NfeR) in E. coli.  J Inorg Biochem. 2018, 184, 123-133.  DOI: 10.1016/j.jinorgbio.2018.04.016.

Dai, Y.; Kim, D.; Dong, G.; Busenlehner, L.S.; Frantom, P.A.; Outten, F.W.  SufE D74R substitution alters active site loop dynamics to further enhance SufE interaction with the SufS Cysteine Desulfurase.  Biochemistry. 2015, 54(31), 4824-4833.        DOI: 10.1021/acs.biochem.5b00663.

Boyd, E. S.; Thomas K. M.; Dai. Y.; Boyd, J. M.; Outten, F. W. Interplay between oxygen and Fe-S cluster biogenesis: insights from the Suf pathway. Biochemistry 2014, 53(37), 5834 - 5847. DOI: 10.1021/bi500488r.

Wang, S.; Blahut, M.; Wu, Y.; Philipkosky, K. E.; Outten, F. W. Communication between binding sites is required for YqjI regulation of target promoters within the yqjH-yqjI intergenic region. J Bacteriol. 2014, 196(17), 3199 - 3207. DOI: 10.1128/JB.01835-14.