Dioxygen and nitric oxide scavenging by Treponema denticola flavodiiron protein: a mechanistic paradigm for catalysis

Abstract

Flavodiiron proteins (FDPs) contain a unique active site consisting of a non-heme diiron carboxylate site proximal to a flavin mononucleotide (FMN). FDPs serve as the terminal components for reductive scavenging of dioxygen (to water) or nitric oxide (to nitrous oxide), which combats oxidative or nitrosative stress in many bacteria. Characterizations of FDPs from spirochetes or from any oral microbes have not been previously reported. Here, we report characterization of an FDP from the anaerobic spirochete, Treponema (T.) denticola, which is associated with chronic periodontitis. The isolated T. denticola FDP exhibited efficient four-electron dioxygen reductase activity and lower but significant anaerobic nitric oxide reductase activity. A mutant T. denticola strain containing the inactivated FDP-encoding gene was significantly more air-sensitive than the wild-type strain. Single turnover reactions of the four-electron-reduced FDP (FMNH2–FeIIFeII) (FDPred) with O2 monitored on the milliseconds to seconds time scale indicated initial rapid formation of a spectral feature consistent with a cis-μ-1,2-peroxo-diferric intermediate, which triggered two-electron oxidation of FMNH2. Reaction of FDPred with NO showed apparent cooperativity between binding of the first and second NO to the diferrous site. The resulting diferrous dinitrosyl complex triggered two-electron oxidation of the FMNH2. Our cumulative results on this and other FDPs indicate that smooth two-electron FMNH2 oxidation triggered by the FDPred/substrate complex and overall four-electron oxidation of FDPred to FDPox constitutes a mechanistic paradigm for both dioxygen and nitric oxide reductase activities of FDPs. Four-electron reductive O2 scavenging by FDPs could contribute to oxidative stress protection in many other oral bacteria.

Spectroscopy and DFT Calculations of Flavo–Diiron Nitric Oxide Reductase Identify Bridging Structures of NO-Coordinated Diiron Intermediates

“…The Dv FDP was expressed from this transformed strain grown as 1 L cultures in Luria-Bertani broth supplemented with ampicillin (100 mg/L) using a previously described protocol. 24 The thawed cell pellets were resuspended in 100 mM MOPS, 500 mM NaCl, and 20 mM imidazole, pH 7.4 (4 mL/g pellet) and sonicated at 4 °C to lyse the cells, and the filtered supernatant from the lysed cells was applied to a Ni Sepharose High Performance resin (GE Healthcare) pre-equilibrated with the resuspension buffer. The column was washed with the same buffer, and the His-tagged Dv FDP was eluted with 5 column volumes of 100 mM MOPS, 500 mM NaCl, and 500 mM imidazole, pH 7.4.…”

The Biologically Relevant Coordination Chemistry of Iron and Nitric Oxide: Electronic Structure and Reactivity

“…Besides nitric oxide dioxygenases (see Section ), pathogens can also express bacterial NORs (see Section ) and non-heme flavodiiron nitric oxide reductases (FNORs) in low oxygen environments . FNORs are scavenging flavodiiron proteins whose gene sequences are found in numerous pathogens, − including Escherichia coli , Salmonella enterica , Treponema denticola , and Desulfovibrio vulgaris . ,− Moreover, the regulator sequences for these FNORs have also been identified in the bacterial pathogens Klebsiella pneumoniae , Vibrio vulnificus , and Salmonella typhimurium . Considering the ability of these pathogens to counteract NO-based immune defense mechanisms , and prolong disease, studying these enzymes is of particular interest.…”

Bioinorganic Chemistry of the Natural [Fe(NO)2] Motif: Evolution of a Functional Model for NO-Related Biomedical Application and Revolutionary Development of a Translational Model

“…To respond to the infections by pathogenic microorganisms, recruitment and activation of host neutrophils and macrophages occurs to produce NO targeting the microbial Fe–S proteins, whereas the microorganisms devise a defense mechanism using the repair of Fe centers (RIC) proteins for the repair of damaged Fe–S clusters and for the removal of NO, presumably through the reduction of NO. , NO reductase utilizes a nonheme carboxylate-bridged diiron active site to catalyze the reductive coupling of two molecules of NO to produce N 2 O (2NO + 2e – + 2H + → N 2 O + H 2 O) in denitrifying bacteria and fungi living in soil and seawater. Regarding the proposed formation of [Fe III -{Fe­(NO) 2 } 9 ] and [Fe III Fe III (hyponitrite)] intermediates, in addition to the proposed intermediates diferrous dinitrosyl [{Fe­(NO)} 7 ] 2 and the super-reduced [{Fe­(NO)} 8 ] 2 species, during the catalytic reaction, NO reduction reactivity was attempted in the synthetic and biomimetic study of {Fe­(NO) 2 } 9 and {Fe­(NO) 2 } 10 DNICs. − The strong antiferromagnetic coupling between the high-spin Fe center and NO ligands forcing parallel spins in the NO ligands within the [Fe­(NO) 2 ] core, however, excluded its reactivity toward N–N bond formation and hyponitrite formation to afford N 2 O . Despite the absence of NO-to-N 2 O reactivity in the [Fe­(NO) 2 ] motif, the research experiences and spectroscopic tools developed during the bioinorganic journey in DNICs inspires our endeavor to seek and unveil control of NO reduction through collaboration on the reactivity and crystallographic investigation of YtfE protein belonging to the RIC family. , …”