Understanding and Exploiting Enzymatic Redox Catalysis

Redox reactions are the foundation of life.  Reactions in which a substrate becomes more oxidized or more reduced are known as ‘redox’ reactions. These are the basis for much of the energy used to support life: reduction of O2 to 2 H2O in respiration, oxidation of sugars to CO2 and countless chemotrophic modes of metabolism. Redox enzymes accelerate these reactions enough for them to be biologically useful and support life (sugar sitting on your table in air does not oxidize significantly in a year, but you get energy from eating, and oxidizing, sugar in a few minutes). Some of the most demanding reactions in biology are catayzed by redox-active enzymes, for example reductive cleavage of the triple bond of N2, which is the second strongest bond known. Yet nitrogenase cleaves N2 at standard temperature and pressure. Many redox-active enzymes use inorganic or organic cofactors to actually execute the chemistry, which in many cases would not be accessible via amino acids alone. However having incorporated reactive cofactors, the protein must be able to control the activated intermediates that occur during turnover, and ensure that only a specific substrate has access to the cofactor, and a single specific reaction follows. Thus, instead of mediating wholesale, random oxidation of all the many susceptible molecules that make up a cell, redox enzymes permit utilization of specific reactions as sources of energy, in parallel with separate utilization of other similar molecules to build and run the cell, and thus provide the basis for controlled burning of selected fuels only, as opposed to an all-consuming wild-fire. 

Enzymes are needed to make redox reactions useful and to control them.  We seek to understand how enzymes can both accelerate difficult reactions by many orders of magnitude (factors of up to x1019), yet on the other hand retain very tight control over what compounds react and what course the reaction takes. These tasks are especially challenging for redox enzymes, since they imply control over the outcome of movements of electrons, and electrons are exceedingly small, fast moving, fundamental particles, while proteins are relatively large molecules, whose structures can be inherently dynamic and flexible. Thus we truly work at the interface between physics and some of the most important questions underlying the possibility of life. Our research addresses fundamental elements of enzymatic redox catalysis, and enzyme catalysis in general.

We are currently concentrating on three enzymes, superoxide dismutase (SOD) and nitroreductase (NR).  These provide complementary perspectives on how redox catalysis can be accomplished, and keep us thinking about how our studies of a particular system can elucidate recurring themes in redox catalysis in general.  Thus, SOD mediates single-electron chemistry based on a bound metal ion (Fe or Mn), and NR exploits an organic cofactor (flavin) in two-electron chemistry. SOD consumes superoxide radicals and thus protects against oxidative damage and forestalls aging. NR metabolizes toxic nitrated aromatics such as TNT and has potential utility in bioremediation, weapons detection and cancer therapy. 

Spectroscopic methods enable us to directly observe the transitions or orbitals of the valence electrons that participate in the reaction. EPR is used to monitor redox-active metal ions directly and thus elucidate the nature of the orbitals accommodating the valence electrons. NMR spectroscopy is used to directly observe the flavin cofactor to elucidate the valence orbitals, and define the geometry and protonation of the system, as well as the hydrogen bonds that tune its reactivity. Since proton transfer is tightly coupled to biological electron transfer, and protons can be directly observed by NMR, NMR is also invaluable for identifying the amino acid residues which participate, and evaluating the energies involved. NMR, EPR and a variety of other spectroscopic methods are used to study substrate binding modes, substrate binding energies, substrate activation and active site residues involved, and NMR enables us to correlate movements throughout the protein with chemical activity of the active site. Our spectroscopic studies are complemented by X-ray crystallography, mechanistic and structural studies, and are aided by the full battery of modern molecular biological methods including site directed mutagenesis, directed evolution, mass spectrometry and specific isotopic labeling. 

Descriptions of specific projects follow.

1          Redox tuning in SOD via a solvent molecule bound to Fe or Mn.

2          Substrate binding to SOD and its relation to electronic configuration and electron transfer.

3          Modification of the active site of SOD to produce a facial triad.

4          Electron redistribution in flavins as a means of protein control over flavin reactivity.

5          Redox tuning in nitroreductase via hydrogen bonds to the flavin.

6          Significance of protein dynamics in the expanded substrate repertoire of nitroreductase.


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last updated: May 2008 comments: send email to AFM 
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