Characterisation of the Cyanate Inhibited State of Cytochrome c Oxidase

Fabian Kruse,Ramona Schlesinger,Joachim Heberle,Anh Duc Nguyen,Inez M Weidinger,Jovan Dragelj,Maria Andrea Mroginski

doi:10.1038/s41598-020-60801-0

Fabian Kruse, Ramona Schlesinger + Show 5 more

Open Access

https://doi.org/10.1038/s41598-020-60801-0

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Abstract

Heme-copper oxygen reductases are terminal respiratory enzymes, catalyzing the reduction of dioxygen to water and the translocation of protons across the membrane. Oxygen consumption is inhibited by various substances. Here we tested the relatively unknown inhibition of cytochrome c oxidase (CcO) with isocyanate. In contrast to other more common inhibitors like cyanide, inhibition with cyanate was accompanied with the rise of a metal to ligand charge transfer (MLCT) band around 638 nm. Increasing the cyanate concentration furthermore caused selective reduction of heme a. The presence of the CT band allowed for the first time to directly monitor the nature of the ligand via surface-enhanced resonance Raman (SERR) spectroscopy. Analysis of isotope sensitive SERR spectra in comparison with Density Functional Theory (DFT) calculations identified not only the cyanate monomer as an inhibiting ligand but suggested also presence of an uretdion ligand formed upon dimerization of two cyanate ions. It is therefore proposed that under high cyanate concentrations the catalytic site of CcO promotes cyanate dimerization. The two excess electrons that are supplied from the uretdion ligand lead to the observed physiologically inverse electron transfer from heme a3 to heme a.

Highlights

Heme-copper oxygen reductases are terminal respiratory enzymes, catalyzing the reduction of dioxygen to water and the translocation of protons across the membrane
A bridging peroxide (O22−) has been proposed to exist in the resting state of CcO1 that requires two more electrons to get fully reduced than the oxidized state during catalytic turnover
In the present work we have investigated the effects of iso-cyanate inhibition via resonance Raman spectroscopy, which is an exquisite method to derived molecular information on the structure of intermediate states of CcO36–38, in combination with quantum chemical calculations[39,40]

Summary

Introduction

Heme-copper oxygen reductases are terminal respiratory enzymes, catalyzing the reduction of dioxygen to water and the translocation of protons across the membrane. Intense research has been performed on cyanide[2,3,4,5,6,7] (CN−) ligands that effectively block the BNC for oxygen While this inhibition of CcO is a central subject in medical research on cyanide poisoning[8,9,10,11], spectroscopic studies on the binding properties of ligands such as cyanide, azide (N3−) or sulphide (S2−)[2,12,13] have been performed to derive the oxygen reduction mechanism, to get insight into the structure of the active site[2,5,6,14] and for spectral isolation of the two heme centers[15,16,17,18]. Immobilization of CcO on plasmonic Ag electrodes offers the exploitation of surface enhancement[41,42], which drastically reduces the amount of protein by several orders of magnitude and allows for electrochemical studies[43,44]

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