Characterization of NosX from Achromobacter cycloclastes and co-crystallization of N 2OR and PAZ

Abstract

Nitrous oxide (N 2O) is known as a greenhouse gas and an ozone-depleting substance. Denitrifying bacteria such as Achromobacter cycloclastes emit N 2O into the atmosphere during the denitrification process. Up to seventy percent of the total N 2O emission on the earth is derived from this process. N 2O is a very stable substance and its lifetime in the atmosphere is 114 years. Investigation of N 2O reductase (N 2OR) and its accessory proteins may ultimately be useful in the design of catalytic systems, either biological or chemical, for the control of N 2O level. The first aim of this research is to characterize NosX, which is one of the accessory proteins for N 2OR. Although NosX was shown to be involved in the activation of N 2OR, the structure and function of NosX and the mechanism of NosX on N 2OR activation are still unknown. Furthermore, NosX has never been isolated and probed. In this study, culture conditions for NosX expression in A. cycloclastes (AcNosX) were optimized to obtain adequate amounts of AcNosX (chapter 2), and AcNosX was characterized with UV/Vis, fluorescence, EPR, and MS spectrometry (chapter 3). The structure of AcNosX was investigated by computational and modeling methods (chapter 3). Crystallization trials were also undertaken. The mechanism of N 2OR activation by AcNosX was studied with UV/Vis and EPR spectrometry (chapter 4). We demonstrated that AcNosX was redox active and donated electrons to the Cu sites of AcN 2OR. The information revealed from this research will help to fully understand the activation mechanism of N 2OR with NosX. The second aim of the research is to investigate the interaction between AcN 2OR and its potential physiological electron donor, pseudoazurin (PAz) (chapter 5). X-ray crystallographic techniques were utilized to determine the structure of the AcN 2ORAcPAz complex and conditions were identified that grew crystals that included both AcN 2OR and AcPAz. Although the crystals showed diffraction patterns, further optimization is required to identify the complex structure. The information from this research will be important to elucidate the docking mechanism for electron transfer between N 2OR and PAz.