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

The conversion of CO by enzymes such as carbonic anhydrase or carboxylases plays a crucial role in many biological processes. However, methods following the microscopic details of CO conversion at the active site are limited. Here, we used infrared spectroscopy to study the interaction of CO, water, bicarbonate, and other reactants with β-carbonic anhydrase from (CA) and crotonyl-CoA carboxylase/reductase from (Ccr), two of the fastest CO-converting enzymes in nature.

Myoglobin-Catalyzed Azide Reduction Proceeds via an Anionic Metal Amide Intermediate.

Nitrene transfer reactions catalyzed by heme proteins have broad potential for the stereoselective formation of carbon-nitrogen bonds. However, competition between productive nitrene transfer and the undesirable reduction of nitrene precursors limits the broad implementation of such biocatalytic methods. Here, we investigated the reduction of azides by the model heme protein myoglobin to gain mechanistic insights into the factors that control the fate of key reaction intermediates.

Enzymatic Conversion of CO: From Natural to Artificial Utilization.

Enzymatic carbon dioxide fixation is one of the most important metabolic reactions as it allows the capture of inorganic carbon from the atmosphere and its conversion into organic biomass. However, due to the often unfavorable thermodynamics and the difficulties associated with the utilization of CO, a gaseous substrate that is found in comparatively low concentrations in the atmosphere, such reactions remain challenging for biotechnological applications. Nature has tackled these problems by evolution of dedicated CO-fixing enzymes, i.

Noncanonical Heme Ligands Steer Carbene Transfer Reactivity in an Artificial Metalloenzyme*.

Changing the primary metal coordination sphere is a powerful strategy for tuning metalloprotein properties. Here we used amber stop codon suppression with engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes, to replace the proximal histidine in myoglobin with N -methylhistidine, 5-thiazoylalanine, 4-thiazoylalanine and 3-(3-thienyl)alanine. In addition to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein's carbene transfer activity with ethyl diazoacetate.

Trapping Transient Protein Species by Genetic Code Expansion.

Nature employs a limited number of genetically encoded amino acids for the construction of functional proteins. By engineering components of the cellular translation machinery, however, it is now possible to genetically encode noncanonical building blocks with tailored electronic and structural properties. The ability to incorporate unique chemical functionality into proteins provides a powerful tool to probe mechanism and create novel function.