Gamma Carbonic Anhydrases from Hydrothermal Vent Bacteria: Cases of Alternating Active Site Due to a Long Loop with Proton Shuttle Residue

Accelerated CO2 sequestration uses carbonic anhydrases (CAs) as catalysts; thus, there is much research on these enzymes. The γ-CA from Escherichia coli (EcoCA-γ) was the first γ-CA to display an active site that switches between “open” and “closed” states through Zn2+ coordination by the proton-shuttling His residue. Here, we explored this occurrence in γ-CAs from hydrothermal vent bacteria and also the γ-CA from Methanosarcina thermophila (Cam) using molecular dynamics. Ten sequences were analyzed through multiple sequence alignment and motif analysis, along with three others from a previous study. Conservation of residues and motifs was high, and phylogeny indicated a close relationship amongst the sequences. All structures, like EcoCA-γ, had a long loop harboring the proton-shuttling residue. Trimeric structures were modeled and simulated for 100 ns at 423 K, with all the structures displaying thermostability. A shift between “open” and “closed” active sites was observed in the 10 models simulated through monitoring the behavior of the His proton-shuttling residue. Cam, which has two Glu proton shuttling residues on long loops (Glu62 and Glu84), also showed an active site switch affected by the first Glu proton shuttle, Glu62. This switch was thus concluded to be common amongst γ-CAs and not an isolated occurrence.

Neutron structures of Leishmania mexicana triosephosphate isomerase in complex with reaction-intermediate mimics shed light on the proton-shuttling steps

Triosephosphate isomerase (TIM) is a key enzyme in glycolysis that catalyses the interconversion of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This simple reaction involves the shuttling of protons mediated by protolysable side chains. The catalytic power of TIM is thought to stem from its ability to facilitate the deprotonation of a carbon next to a carbonyl group to generate an enediolate intermediate. The enediolate intermediate is believed to be mimicked by the inhibitor 2-phosphoglycolate (PGA) and the subsequent enediol intermediate by phosphoglycolohydroxamate (PGH). Here, neutron structures of Leishmania mexicana TIM have been determined with both inhibitors, and joint neutron/X-ray refinement followed by quantum refinement has been performed. The structures show that in the PGA complex the postulated general base Glu167 is protonated, while in the PGH complex it remains deprotonated. The deuteron is clearly localized on Glu167 in the PGA–TIM structure, suggesting an asymmetric hydrogen bond instead of a low-barrier hydrogen bond. The full picture of the active-site protonation states allowed an investigation of the reaction mechanism using density-functional theory calculations.

In Silico Investigation of Potential Applications of Gamma Carbonic Anhydrases as Catalysts of CO2 Biomineralization Processes: A Visit to the Thermophilic Bacteria Persephonella hydrogeniphila, Persephonella marina, Thermosulfidibacter takaii, and Thermus thermophilus

Carbonic anhydrases (CAs) have been identified as ideal catalysts for CO2 sequestration. Here, we report the sequence and structural analyses as well as the molecular dynamics (MD) simulations of four γ-CAs from thermophilic bacteria. Three of these, Persephonella marina, Persephonella hydrogeniphila, and Thermosulfidibacter takaii originate from hydrothermal vents and one, Thermus thermophilus HB8, from hot springs. Protein sequences were retrieved and aligned with previously characterized γ-CAs, revealing differences in the catalytic pocket residues. Further analysis of the structures following homology modeling revealed a hydrophobic patch in the catalytic pocket, presumed important for CO2 binding. Monitoring of proton shuttling residue His69 (P. marina γ-CA numbering) during MD simulations of P. hydrogeniphila and P. marina’s γ-CAs (γ-PhCA and γ-PmCA), showed a different behavior to that observed in the γ-CA of Escherichia coli, which periodically coordinates Zn2+. This work also involved the search for hotspot residues that contribute to interface stability. Some of these residues were further identified as key in protein communication via betweenness centrality metric of dynamic residue network analysis. T. takaii’s γ-CA showed marginally lower thermostability compared to the other three γ-CA proteins with an increase in conformations visited at high temperatures being observed. Hydrogen bond analysis revealed important interactions, some unique and others common in all γ-CAs, which contribute to interface formation and thermostability. The seemingly thermostable γ-CA from T. thermophilus strangely showed increased unsynchronized residue motions at 423 K. γ-PhCA and γ-PmCA were, however, preliminarily considered suitable as prospective thermostable CO2 sequestration agents.

The mitochondria-targeted derivative of the classical uncoupler of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone is an effective mitochondrial recoupler

The synthesis of a mitochondria-targeted derivative of the classical mitochondrial uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP) by alkoxy substitution of CCCP with n-decyl(triphenyl)phosphonium cation yielded mitoCCCP, which was able to inhibit the uncoupling action of CCCP, tyrphostin A9 and niclosamide on rat liver mitochondria, but not that of 2,4-dinitrophenol, at a concentration of 1–2 μM. MitoCCCP did not uncouple mitochondria by itself at these concentrations, although it exhibited uncoupling action at tens of micromolar concentrations. Thus, mitoCCCP appeared to be a more effective mitochondrial recoupler than 6-ketocholestanol. Both mitoCCCP and 6-ketocholestanol did not inhibit the protonophoric activity of CCCP in artificial bilayer lipid membranes, which might compromise the simple proton-shuttling mechanism of the uncoupling activity on mitochondria.

Ni-Catalyzed asymmetric reduction of α-keto-β-lactams via DKR enabled by proton shuttling

A wide range of α-keto-β-lactams were reduced efficiently and enantioselectively by Ni-catalyzed asymmetric hydrogenation. Phenylphosphinic acid was found to play a pivotal role in the DKR of α-keto-β-lactams by promoting the enolization process.

Metal vs. ligand protonation and the alleged proton-shuttling role of the azadithiolate ligand in catalytic H2 formation with FeFe hydrogenase model complexes

Real-time spectroscopic observation of electron transfer-induced protonation reactivity elucidates the role of the second sphere basic site in a H2 evolution catalyst.