Infrared spectroscopy reveals metal-independent carbonic anhydrase activity in crotonyl-CoA carboxylase/reductase.

The conversion of CO2 by enzymes such as carbonic anhydrase or carboxylases plays a crucial role in many biological processes. However, in situ methods following the microscopic details of CO2 conversion at the active site are limited. Here, we used infrared spectroscopy to study the interaction of CO2, water, bicarbonate, and other reactants with ß-carbonic anhydrase from Escherichia coli (EcCA) and crotonyl-CoA carboxylase/reductase from Kitasatospora setae (KsCcr), two of the fastest CO2-converting enzymes in nature. Our data reveal that KsCcr possesses a so far unknown metal-independent CA-like activity. Site-directed mutagenesis of conserved active site residues combined with molecular dynamics simulations tracing CO2 distributions in the active site of KsCCr identify an 'activated' water molecule forming the hydroxyl anion that attacks CO2 and yields bicarbonate (HCO3-). Computer simulations also explain why substrate binding inhibits the anhydrase activity. Altogether, we demonstrate how in situ infrared spectroscopy combined with molecular dynamics simulations provides a simple yet powerful new approach to investigate the atomistic reaction mechanisms of different enzymes with CO2.

Conformational Dynamics of the Most Efficient Carboxylase Contributes to Efficient CO2 Fixation.

Crotonyl-CoA carboxylase/reductase (Ccr) is one of the fastest CO2 fixing enzymes and has become part of efficient artificial CO2-fixation pathways in vitro, paving the way for future applications. The underlying mechanism of its efficiency, however, is not yet completely understood. X-ray structures of different intermediates in the catalytic cycle reveal tetramers in a dimer of dimers configuration with two open and two closed active sites. Upon binding a substrate, this active site changes its conformation from the open state to the closed state. It is challenging to predict how these coupled conformational changes will alter the CO2 binding affinity to the reaction's active site. To determine whether the open or closed conformations of Ccr affect binding of CO2 to the active site, we performed all-atom molecular simulations of the various conformations of Ccr. The open conformation without a substrate showed the highest binding affinity. The CO2 binding sites are located near the catalytic relevant Asn81 and His365 residues and in an optimal position for CO2 fixation. Furthermore, they are unaffected by substrate binding, and CO2 molecules stay in these binding sites for a longer time. Longer times at these reactive binding sites facilitate CO2 fixation through the nucleophilic attack of the reactive enolate in the closed conformation. We previously demonstrated that the Asn81Leu variant cannot fix CO2. Simulations of the Asn81Leu variant explain the loss of activity through the removal of the Asn81 and His365 binding sites. Overall, our findings show that the conformational dynamics of the enzyme controls CO2 binding. Conformational changes in Ccr increase the level of CO2 in the open subunit before the substrate is bound, the active site closes, and the reaction starts. The full catalytic Ccr cycle alternates among CO2 addition, conformational change, and chemical reaction in the four subunits of the tetramer coordinated by communication between the two dimers.

Intersubunit Coupling Enables Fast CO2-Fixation by Reductive Carboxylases.

Enoyl-CoA carboxylases/reductases (ECRs) are some of the most efficient CO2-fixing enzymes described to date. However, the molecular mechanisms underlying the extraordinary catalytic activity of ECRs on the level of the protein assembly remain elusive. Here we used a combination of ambient-temperature X-ray free electron laser (XFEL) and cryogenic synchrotron experiments to study the structural organization of the ECR from Kitasatospora setae. The K. setae ECR is a homotetramer that differentiates into a pair of dimers of open- and closed-form subunits in the catalytically active state. Using molecular dynamics simulations and structure-based mutagenesis, we show that catalysis is synchronized in the K. setae ECR across the pair of dimers. This conformational coupling of catalytic domains is conferred by individual amino acids to achieve high CO2-fixation rates. Our results provide unprecedented insights into the dynamic organization and synchronized inter- and intrasubunit communications of this remarkably efficient CO2-fixing enzyme during catalysis.

Conformational sampling and polarization of Asp26 in pKa calculations of thioredoxin.

Thioredoxin is a protein that has been used as model system by various computational methods to predict the pKa of aspartate residue Asp26 which is 3.5 units higher than a solvent exposed one (eg, Asp20). Here, we use extensive atomistic molecular dynamics simulations of two different protonation states of Asp26 in combination with conformational analysis based on RMSD clustering and principle component analysis to identify representative conformations of the protein in solution. For each conformation, the Gibbs free energy of proton transfer between Asp26 and Asp20, which is fully solvated in a loop region of the protein, is calculated with the Amber99sb force field in alchemical transformations. The varying polarization of the two residues in different molecular environments and protonation states is described by Hirshfeld-I (HI) atomic charges obtained from the averaged polarized electron density. Our results show that the Gibbs free energy of proton transfer is dependent on the protein conformation, the proper sampling of the neighboring Lys57 residue orientations and on water molecules entering the hydrophobic cavity upon deprotonating Asp26. The inclusion of the polarization of both aspartate residues in the free energy cycle by HI atomic charges corrects the results from the non-polarizable force field and reproduces the experimental ΔpKa value of Asp26.