Structure-function relationships in metalloenzymes with multiple redox-active centers

With the support of the Chemistry of Life Processes program in the Division of Chemistry, Professor A. Andrew Pacheco of the University of Wisconsin-Milwaukee studies the chemistry of biological interconversion between ammonia and nitrite. Ammonia, which is a major component of fertilizers, and nitrite are two examples of “reactive nitrogen” that is, nitrogen usable by many living organisms, as opposed to “elemental nitrogen”, which makes up 78% of the air we breathe but is directly usable by only a few bacteria.

Over the last 50-years the balance between reactive and elemental nitrogen has shifted significantly towards the former, as more fertilizer was generated to produce food and (recently) biofuels. This shift has many unintended negative consequences, which will soon have to be mitigated. A better understanding of ammonia-nitrite interconversion may lead to the more efficient use of ammonia fertilizer, and thus help redress the imbalance.

The project’s highly interdisciplinary nature will provide the graduate, undergraduate and high school students who conduct the research with a wide breadth of skills that will make them very competitive in their independent careers. The high school students will be recruited through Summer Experiences for the Economically Disadvantaged Program of the American Chemical Society.

The proposed research will concentrate on the reaction mechanism of cytochrome-c nitrite reductase (ccNIR), an enzyme that allows certain bacteria to reduce nitrite to ammonia. The bacteria can extract energy from the process; in the absence of ccNiR, this process would be too slow for bacterial survival. Previous studies by the Pacheco group showed that putative catalytic intermediates can be trapped and studied when weak reductants are used as electron sources.

This strategy will be used to trap putative catalytic intermediates of nitrite-loaded ccNiR in which the active site is 1-electron, 2-electron and 4-electron reduced relative to the resting state of the enzyme. Kinetic studies using UV/Vis stopped-flow will be used to determine the timescales on which intermediates accumulate, after which samples of these intermediates will be prepared by rapid freeze-quench and investigated using a variety of electron paramagnetic resonance spectroscopic methods (CW-EPR and pulsed-EPR) and Mossbauer spectroscopy.

The primary goal of these studies is to determine how the ccNiR active site is optimized to resist releasing partially-reduced nitrogenous intermediates, such as nitric oxide, nitrous oxide or hydroxylamine, during catalysis.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.