A Conformational Switch Triggers Nitrogenase Protection from Oxygen Damage by Shethna Protein II (FeSII)

Abstract

The two-component metalloprotein nitrogenase catalyzes the reductive fixation of atmospheric dinitrogen into bioavailable ammonium in diazotrophic prokaryotes. The process requires an efficient energy metabolism, so that although the metal clusters of nitrogenase rapidly decompose in the presence of dioxygen, many free-living diazotrophs are obligate aerobes. In order to retain the functionality of the nitrogen-fixing enzyme, some of these are able to rapidly "switch-off" nitrogenase, by shifting the enzyme into an inactive but oxygen-tolerant state. Under these conditions the two components of nitrogenase form a stable, ternary complex with a small [2Fe:2S] ferredoxin termed FeSII or the "Shethna protein II". Here we have produced and isolated Azotobacter vinelandii FeS II and have determined its three-dimensional structure to 2.1 Å resolution by X-ray diffraction. In the crystals, the dimeric protein was present in two distinct states that differ in the conformation of an extended loop in close proximity to the iron-sulfur cluster. We show that this rearrangement is redox-dependent and forms the molecular basis for oxygen-dependent conformational protection of nitrogenase. Protection assays highlight that FeSII binds to a preformed complex of MoFe and Fe protein upon activation, primarily through electrostatic interactions. The surface properties and known complexes of nitrogenase component proteins allow us to propose a model of the conformationally protected ternary complex of nitrogenase.

Where in the world do bacteria experience oxidative stress?

“…Oxygen forms adducts to glycylradical enzymes, leading to polypeptide cleavage (Wagner et al, 1992) of enzymes such as pyruvate:formate lyase and ribonucleotide reductase. Further, it seems likely that molecular oxygen itself can over-oxidize the low-potential metal centres of enzymes such as pyruvate:ferredoxin oxidoreductase, hydrogenase and nitrogenase (Vita et al, 2008;Stiebritz and Reiher, 2012;Schlesier et al, 2015). These enzymes are rapidly damaged by the introduction of oxygen in vitro and in vivo.…”

Combinatorial design of chemical‐dependent protein switches for controlling intracellular electron transfer

“…Several library approaches have been described for generating domain insertion libraries, including nuclease‐based and transposase‐mediated methods . Among these approaches, transposase mutagenesis is appealing to use for creating protein switches because it avoids deletions that can arise with nuclease‐based methods . Transposase mutagenesis has recently been used to discover chemical‐dependent protein switches by targeting lactamase, CRISPR‐Cas9, green fluorescent protein (GFP), and an amino‐acyl tRNA synthetase for random domain insertion.…”

“…Recently, evidence emerged that Fds may at times use post‐translational control to regulate ET in cells. Nitrogenase‐protecting Fds use oxidation to alter their conformation and binding to nitrogenase as a mechanism to protect their partner protein from oxidative damage . In addition, phosphorylated and calcium‐bound Fds have been reported, suggesting that fast regulation of ET has arisen during evolution .…”

Dinitrogen Reduction: Interfacing the Enzyme Nitrogenase with Electrodes

“…As a final perspective, we note that a central challenge regarding the biological reduction of N 2 is that both of the Fe and MoFe proteins that compose the nitrogenase enzyme are irreversibly damaged by oxygen. The former is particularly sensitive . Nitrogenase‐based photobiological production of H 2 is possible in aerobic photosynthetic microorganisms such as cyanobacteria on condition that H 2 production is temporally or spatially separated from photosynthesis, and oxygen sensitivity of nitrogenase has been recognized as an obstacle for bioengineering nitrogen fixation into cereal crops.…”