Evidence of Atypical Structural Flexibility of the Active Site Surrounding of an [FeFe] Hydrogenase from Clostridium beijerinkii.

[FeFe] hydrogenase from Clostridium beijerinkii (CbHydA1) is an unusual hydrogenase in that it can withstand prolonged exposure to O2 by reversibly converting into an O2-protected, inactive state (Hinact). It has been indicated in the past that an atypical conformation of the "SC367CP" loop near the [2Fe]H portion of the six-iron active site (H-cluster) allows the Cys367 residue to adopt an "off-H+-pathway" orientation, promoting a facile transition of the cofactor to Hinact. Here, we investigated the electronic structure of the H-cluster in the oxidized state (Hox) that directly converts to Hinact under oxidizing conditions and the related CO-inhibited state (Hox-CO). We demonstrate that both states exhibit two distinct forms in electron paramagnetic resonance (EPR) spectroscopy. The ratio between the two forms is pH-dependent but also sensitive to the buffer choice. Our IR and EPR analyses illustrate that the spectral heterogeneity is due to a perturbation of the coordination environment of the H-cluster's [4Fe4S]H subcluster without affecting the [2Fe]H subcluster. Overall, we conclude that the observation of two spectral components per state is evidence of heterogeneity of the environment of the H-cluster likely associated with conformational mobility of the SCCP loop. Such flexibility may allow Cys367 to switch rapidly between off- and on-H+-pathway rotamers. Consequently, we believe such structural mobility may be the key to maintaining high enzymatic activity while allowing a facile transition to the O2-protected state.

Investigation of the Unusual Ability of the [FeFe] Hydrogenase from Clostridium beijerinckii to Access an O2-Protected State.

[FeFe] hydrogenases are enzymes capable of producing and oxidizing H2 at staggering submillisecond time scales. A major limitation in applying these enzymes for industrial hydrogen production is their irreversible inactivation by oxygen. Recently, an [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1) was reported to regain its catalytic activity after exposure to oxygen. In this report, we have determined that artificially matured CbHydA1 is indeed oxygen tolerant in the absence of reducing agents and sulfides by means of reaching an O2-protected state (Hinact). We were also able to generate the Hinact state anaerobically via both chemical and electrochemical oxidation. We use a combination of spectroscopy, electrochemistry, and density functional theory (DFT) to uncover intrinsic properties of the active center of CbHydA1, leading to its unprecedented oxygen tolerance. We have observed that reversible, low-potential oxidation of the active center leads to the protection against O2-induced degradation. The transition between the active oxidized state (Hox) and the Hinact state appears to proceed without any detectable intermediates. We found that the Hinact state is stable for more than 40 h in air, highlighting the remarkable resilience of CbHydA1 to oxygen. Using a combination of DFT and FTIR, we also provide a hypothesis for the chemical identity of the Hinact state. These results demonstrate that CbHydA1 has remarkable stability in the presence of oxygen, which will drive future efforts to engineer more robust catalysts for biofuel production.

Analysis of RNA Methylation by Phylogenetically Diverse Cfr Radical S-Adenosylmethionine Enzymes Reveals an Iron-Binding Accessory Domain in a Clostridial Enzyme.

Cfr is a radical S-adenosylmethionine (SAM) RNA methylase linked to multidrug antibiotic resistance in bacterial pathogens. It catalyzes a chemically challenging C-C bond-forming reaction to methylate C8 of A2503 (Escherichia coli numbering) of 23S rRNA during ribosome assembly. The cfr gene has been identified as a mobile genetic element in diverse bacteria and in the genome of select Bacillales and Clostridiales species. Despite the importance of Cfr, few representatives have been purified and characterized in vitro. Here we show that Cfr homologues from Bacillus amyloliquefaciens, Enterococcus faecalis, Paenibacillus lautus, and Clostridioides difficile act as C8 adenine RNA methylases in biochemical assays. C. difficile Cfr contains an additional Cys-rich C-terminal domain that binds a mononuclear Fe2+ ion in a rubredoxin-type Cys4 motif. The C-terminal domain can be truncated with minimal impact on C. difficile Cfr activity, but the rate of turnover is decreased upon disruption of the Fe2+-binding site by Zn2+ substitution or ligand mutation. These findings indicate an important purpose for the observed C-terminal iron in the native fusion protein. Bioinformatic analysis of the C. difficile Cfr Cys-rich domain shows that it is widespread (∼1400 homologues) as a stand-alone gene in pathogenic or commensal Bacilli and Clostridia, with >10% encoded adjacent to a predicted radical SAM RNA methylase. Although the domain is not essential for in vitro C. difficile Cfr activity, the genomic co-occurrence and high abundance in the human microbiome suggest a possible functional role for a specialized rubredoxin in certain radical SAM RNA methylases that are relevant to human health.