In Situ Infrared Spectroscopy for the Analysis of Gas-processing Metalloenzymes

Abstract

Earth-abundant transition metals, such as iron, nickel, copper, molybdenum, and vanadium, have been identified as essential constituents of the cellular gas metabolism in all kingdoms of life. Associated with biological macromolecules, gas-processing metalloenzymes (GPMs) are formed that catalyze a variety of redox reactions. This includes the reduction of O2 to water by cytochrome c oxidase (“complex IV”), the reduction of N2 to NH3 by nitrogenase, as well as the reversible reduction of protons to H2 by hydrogenase. GPMs perform at ambient temperature and pressure, in the presence of water, and often extremely low educt concentrations, thus serving as natural examples for efficient catalysis. Facilitating the design of biomimetic catalysts, biophysicist thrive to understand the reaction principles of GPMs making use of various techniques. In this Perspective, I will introduce Fourier-transform infrared spectroscopy in attenuated total reflection configuration (ATR FTIR) for the analysis of GPMs like cytochrome c oxidase, nitrogenase, and hydrogenase. Infrared spectroscopy provides information about the geometry and redox state of the catalytic cofactors, the protonation state of amino acid residues, the hydrogen-bonding network, and protein structural changes. I developed an approach to probe and trigger the reaction of GPMs by gas exchange and deuteration experiments exploring the reactivity of these enzymes with their natural reactants. This allows recording sensitive steady-state ATR FTIR (difference) spectra with seconds time resolution. Finally yet importantly, infrared spectroscopy is an electronically noninvasive technique that allows investigating protein samples under biologically relevant conditions, that is, at ambient temperature, ambient pressure, and in the presence of liquid water.

Mechanism of Hydrogen Sulfide-Dependent Inhibition of FeFe Hydrogenase

“…Of course, this mechanism cannot apply in the case of the enzyme from C. reinhardtii, but we show that reactivation can actually occur after the H-cluster in the H trans state is one-electron reduced; the final product, H ox , is still isoelectronic with H trans because of either the potentiometric reoxidation or the auto-oxidation of the reduced states of the H-cluster in the absence of H 2 (as reported before by Stripp et al ,, ). We consider likely that this mechanism also applies in the case ofD.…”

“…suggested that, at least in the case of the enzyme from D. desulfuricans, the reactivation of H trans is triggered by the reduction of an accessory cluster, which induces H 2 S release from the distal Fe by an anti-cooperative, long distance interaction with the H-cluster. Of course this mechanism cannot apply in the case of the enzyme from C. reinhardtii, but we show that reactivation can actually occur after the H trans state is one-electron reduced; the final product is still isoelectronic with H trans , because of either the potentiometric re-oxidation or the auto-oxidation of the reduced states of the H-cluster in the absence of H 2 (as reported before by Stripp et al 14,74,75 ). We consider likely that this simple mechanism also applies in the case of D. desulfuricans hydrogenase.…”

“…In addition to electrochemical reoxidation, catalytic turnover also interconverts H red and H ox ; that this conversion is out of equilibrium invalidates the above argument about the thermodynamics of the binding of H 2 S to the H-cluster (which suggested that H red , rather than H ox , should be the product of the reductive reactivation of H trans ). Catalytic interconversion between H red and H ox occurs in the electrochemical experiments reported here, but also in an FTIR cell: indeed, it has been observed that H red and H red H + are spontaneously auto-oxidized to H ox by catalytic turnover in the absence of H 2 . ,, Auto-oxidation of H red should involve disproportionation, which is probably easy in the FTIR cell where the enzyme concentration is very large and bimolecular reactions should be fast. Whichever mechanism we consider, the spontaneous reoxidation of H red or H red H + simply solves the paradox: reactivation follows up the reduction of H trans , but the final product is isoelectronic with H trans .…”

“…Catalytic interconversion between H red and H ox occurs in the electrochemical experiments reported here, but also in a FTIR cell: indeed, it has been observed that H red and H red H + are spontaneously auto-oxidized to H ox by catalytic turnover in the absence of H 2 . 14,74,75 Auto-oxidation of H red should involve disproportionation, which is probably easy in the FTIR cell where the enzyme concentration is very large and bimolecular reactions should be fast. Whichever mechanism we consider, the spontaneous re-oxidation of H red or H red H + simply solves the paradox: reactivation follows up the reduction of H trans , but the final product is isoelectronic with H trans .…”

Membrane Protein Activity Induces Specific Molecular Changes in Nanodiscs Monitored by FTIR Difference Spectroscopy

“…Our setup ( Senger et al, 2018 ) allowed the rapid exchange of the atmosphere over the protein film with 40% O 2 , triggering the oxidation of Rs C c O. N 2 was used as a carrier over the whole experiment. Every difference spectrum is the result of a different number of co-additions of pre-trigger and post-trigger absolute spectra that maximized SNR ( Stripp, 2021 ), ranging from a minimum of 20 scans to a maximum of 500.…”