Biochemical characterization of cytochrome c2 and nitrite reductase from Neisseria gonorrhoeae

Neisseria gonorrhoeae is the obligate human pathogen responsible for the sexually transmitted disease gonorrhea. The O2-limited environment of its infection site and induced-oxidative stress with ROS, like H2O2, by the immune system creates the necessity of a pathway for anaerobic growth and a peroxide detoxification system for N. gonorrhoeae survival in the human host. The periplasmatic outer membrane-bound enzymes, copper nitrite reductase (NgAniA) and cytochrome c peroxidase (NgCCP), have been associated with anaerobic growth and hydrogen peroxide detoxification, respectively. Cytochrome c2 (NgCytc2), a periplasmic c-type cytochrome, was proposed to act as an electron shuttle between Complex IV of aerobic respiration and NgAniA. Due to its cellular location, it is proposed that it can also transfer electrons to NgCCP. The lipid-modified azurin (NgLaz), a periplasmatic outer membrane-bound type I copper protein and physiological donor of NgCCP, is also a plausible electron donor to NgAniA due to its cellular localization and since azurins commonly donate electrons to CuNiRs. NgCytc2 and NgAniA were heterologously produced in E.coli BL21(DE3) and isolated with two chromatographic steps, an affinity chromatography followed by an cationic exchange chromatography for NgCytc2 and an anionic exchange chromatography for NgAniA, with a yield of 2.5 mg/L and 50 mg/L of culture medium, respectively. The isolated NgCytc2 had one c-type heme per protein, as expected. The UV-visible spectra of NgCytc2 show the Soret band at 410 nm (111 mM-1.cm-1) that shifts to 416 nm (136 mM-1.cm-1) when reduced. The α- and β- bands become evident in the reduced spectrum at 552 nm (20 mM-1.cm-1) and 522 nm (15 mM-1.cm-1), respectively. Two other absorption bands are observed, at 690 nm indicative of a methionine-heme coordination and at 640 nm indictive of a high-spin/low-spin equilibrium a room temperature. The reduction potential of NgCytc2 c-type heme was determined by cyclic voltammetry in an in-solution configuration to be 149 ± 5 mV, vs SHE, pH 7.0. NgAniA was isolated with 1.9 ± 0.1 copper per monomer and its UV-visible spectrum in the oxidized form has two absorption bands at 458 nm (2.1 ± 0.3 mM-1.cm-1) and 598 nm (4.0 ± 0.2 mM-1.cm-1), with an Abs 458 nm/Abs 598 nm of 0.64, indicating that it belongs to the blueish-green copper nitrite reductases. The nitrite reductase activity of NgAniA was assessed with the reduced form of three artificial electron donors: methyl viologen (MV), 1,2-napthoquinone-4-sulfonate (NQS) and 2,6-dichlorophenolindoplenol (DCPIP), with only the first two being able to donate electrons to NgAniA. These assays indicated that the isolated NgAniA was catalytically active, and a specific activity of 109 ± 6 μmol/min/mg and 3.8 ± 0.3 μmol/min/mg was estimated using MV and NQS as electron donors, respectively. NgLaz does not seem to transfer electrons to NgAniA, as NgLaz oxidation was not observed in the presence of NgAniA