A Reduced F ₄₂₀ -Dependent Nitrite Reductase in an Anaerobic Methanotrophic Archaeon

Abstract

Coenzyme F 420 -dependent sulfite reductase (Fsr) protects methanogenic archaea inhabiting deep-sea hydrothermal vents from the inactivation of methyl coenzyme M reductase (Mcr), one of their essential energy production enzymes. Anaerobic methanotrophic archaea (ANME) that oxidize methane and rely on Mcr, carry Fsr homologs that form a distinct clade.

Structures of the sulfite detoxifying F420-dependent enzyme from Methanococcales

“…1) 15 . In the absence of DsrC, DsrAB releases some S 2− , as well as the reaction intermediates trithionate and thiosulfate [15][16][17] .…”

Nitrite reductase activity in F420-dependent sulphite reductase (Fsr) from Methanocaldococcus jannaschii

“…The Arg 98 , Arg 170 , Lys 211 and Lys 213 of Af Dsr are fully conserved in Mj FsrI (Arg 355 , Arg 423 , Lys 460 and Lys 462 [15, 31]) and therefore Mj FsrI employed a common chemical mechanism for reducing sulphite and nitrite; the recently available crystal structure of Mj FsrI indeed shows that Arg 355 , Arg 423 , Lys 460 and Lys 462 are involved in the binding of sulphite and two water molecules at the active site of this protein [31]. The ability of Mj FsrI to reduce hydroxylamine (NH 2 OH) indicated that the Fsr-catalysed reduction of nitrite proceeded through the intermediate formation of hydroxylamine and this sequence is also seen with the other sulphite/nitrite reductases [17, 18, 20, 21]. The enzyme was efficient in the utilization of the F 420 H 2 -derived reducing equivalents as it exhibited almost 100 % recovery of this resource into ammonia, the product.…”

“…The assays for FNiR, Fsr and F 420 H 2 -dependent hydroxylamine reductase were performed anaerobically using reduced F 420 (F 420 H 2 ) as the electron donor and respective electron acceptors, which were nitrite, sulphite and hydroxylamine, following a previously described procedure [15,17,27]. Briefly, this procedure involved spectrophotometric monitoring of the oxidation of F 420 H 2 at 400 nm, and an extinction coefficient value of 25 mM −1 cm −1 for F 420 was used to calculate the reaction rate [28].…”

“…The contents of iron and acid-labile sulphur of MjFsrI were determined as described previously [17], except a solution containing 71.8 µg ml −1 protein in 25 mM potassium phosphate buffer, pH 7, was used. The ammonia produced in the Fsr reaction was estimated using a glutamate dehydrogenase-based assay [15,17,29] employing an AA0100 Kit (Sigma-Aldrich).…”

“…Most sulphite reductases reduce nitrite [15, 17–22] and a preliminary study in our laboratory has shown an indication for this activity in Mj FsrI [23]. Also, recently an FsrII from one of the anaerobic methanotrophic archaea (ANME), which oxidize methane anaerobically, has been shown to act as an F 420 -dependent nitrite reductase (FNiR) and lacks sulphite reductase activity [17]. Accordingly, we examined the nitrite reductase activity of Mj FsrI and characterized the respective kinetic properties.…”