Structure-Function Studies of Nitrate Reductase Enzymes

Abstract

Nitrate reductase enzymes are a class of molybdenum-based enzymes that undergo a 2-electron redox reaction to reduce nitrate (NO3-) to nitrite (NO2-). This class of enzyme is very important in various geothermal cycles, the most prominent of which is the global nitrogen cycle. There are several types of nitrate reductase based on mechanism and overall structure from organisms such as eukaryotes to bacteria. The focus of this study is the periplasmic nitrate reductase from Campylobacter jejuni and the assimilatory nitrate reductase from Synechococcus elongatus. Both share identical primary coordination spheres at the catalytically essential molybdenum and are predicted to share an overall peptide fold motif. Importantly, for these enzymes, the primary coordination sphere and mechanism are open questions in the community and a focus of this work. Through a series of activity assays and analysis using electron paramagnetic resonance (EPR) spectroscopy, the kinetics, and reactivity of the nitrate reductases were probed in the hope of gaining a better understanding of what affects the reactivity of the Mo center, and what might potentially inhibit the enzyme\u27s activity. Kinetic activity assays using methyl viologen (MV) as an electron donator promoted the turnover of nitrate to nitrate, which allowed for the analysis of active enzymes to obtain the specific activity for both types of nitrate reductase. This assay proved that the synthesis methods of both enzymes were successful in producing active enzymes that could be used in further analysis, and also introduced the possibility that dithionite (DT) is not a good electron donator for these enzymes, but an inhibitor to turnover. Through EPR analysis of NapA, the mechanism behind the conversion of NO3- to NO2- was studied. WT CjNapA underwent turnover and the EPR was obtained before and after, which showed a 6-coordinate structure arising after turnover. This structure aligns with a “Sulfur-shift reaction”, that has been recently proposed in opposition to the previously stated “Oxygen-atom reaction”. This observation gives much more insight into not only the mechanism but also the binding pocket and potential reactivity of these types of enzymes. In addition to understanding the mechanism and reactivity of the enzymes, a new equation to help classify them was formed. This new “Rhombicity” equation was tested for a variety of different molybdenum enzymes, as well as for systems with different transition metals than Mo. This equation helps to classify and quantify the degree of separation of EPR spectra, which allows for better analysis of structure

Similar works

Full text

thumbnail-image

University of Wisconsin-Milwaukee

redirect
Last time updated on 19/07/2023

This paper was published in University of Wisconsin-Milwaukee.

Having an issue?

Is data on this page outdated, violates copyrights or anything else? Report the problem now and we will take corresponding actions after reviewing your request.