Exploring the Molecular Machinery of Denitrification in Haloferax mediterranei Through Proteomics

Abstract

Many proteins and enzymes involved in denitrification in haloarchaea can be inferred to be located between the cytoplasmic membrane and the S-layer, based on the presence of a Tat signal sequence and the orientation of the active site that some of these enzymes have. The membrane fraction of the haloarchaeon Haloferax mediterranei (R-4), grown under anaerobic conditions in the presence of nitrate, was solubilized to identify the respiratory proteins associated or anchored to it. Using Triton X-100, CHAPS, and n-Octyl-β-d-glucopyranoside at different concentrations we found the best conditions for isolating membrane proteins in micelles, in which enzymatic activity and stability were maintained. Then, they were subjected to purification using two chromatographic steps followed by the analysis of the eluents by NANO-ESI Chip-HPLC-MS/MS. The results showed that the four main enzymes of denitrification (nitrate, nitrite, nitric oxide, and nitrous oxide reductases) in H. mediterranei were identified and they were co-purified thanks to the micelles made with Triton X-100 (20% w/v for membrane solubilisation and 0.2% w/v in the buffers used during purification). In addition, several accessory proteins involved in electron transfer processes during anaerobic respiration as well as proteins supporting ATP synthesis, redox balancing and oxygen sensing were detected. This is the first characterization of anaerobic membrane proteome of haloarchaea under denitrifying conditions using liquid chromatography-mass spectrometry. It provides new information for a better understanding of the anaerobic respiration in haloarchaea.

Keywords: Haloferax; anaerobiosis; denitrification; detergents; electron transfer; haloarchaea; liquid chromatography-mass spectrometry; proteomics.

Figure 1

Spectral intensity (A), protein sequence coverage (B) adn specific Nar and Nir activities (C) data in the three steps of enrichment. They are shown as the average of triplicates with standard deviation (as bar error).

Figure 2

Number of proteins with different and unique peptides in the micelles during the enrichment process: crude extract (A), DEAE-Sepharose (B), and Q-Sepharose (C). Positive identifications are considered only when two or more peptides are matched, and their summed score is >30.

Figure 3

Schematic model of the potential denitrification super-complex in Haloferax mediterranei. The different reactions catalyzed by the four enzymes (Nitrate-Nar, Nitrite-Nir, Nitric oxide-Nor and Nitrous oxide- Nos reductases) have been represented stoichiometrically. *The number of NO molecules generated by the reduction of one molecule of ${\text{NO}}_2^{-}$ has been multiplied by two to maintain the stoichiometry of the generation of one molecule of N₂O.The red lines represent physiologically tested (continuous lines) or potential(discontinuous lines) electron flows.