SERR Spectroelectrochemical Study of Cytochrome cd1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c552 on Biocompatible Metal Electrodes.

Célia M Silveira,Pedro O Quintas,M Gabriela Almeida,Isabel Moura,Smilja Todorovic,Peter Hildebrandt,José J G Moura,Nikolai Lebedev

doi:10.1371/journal.pone.0129940

Abstract

Cytochrome cd1 nitrite reductases (cd 1NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd 1NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd 1NiR from Marinobacter hydrocarbonoclasticus (Mhcd 1) depends on the presence of its physiological redox partner, cytochrome c 552 (cyt c 552), we show here a detailed surface enhanced resonance Raman characterization of Mhcd 1 and cyt c 552 attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd 1 and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd 1 // cyt c 552 and Ag // SAM // cyt c 552 // Mhcd 1 constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c 552 retains its native properties, while the redox potential of apparently intact Mhcd 1 undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd 1, reinforcing the idea that subtle and very specific interactions between Mhcd 1 and cyt c 552 govern efficient intermolecular electron transfer and catalytic activity of Mhcd 1.

Highlights

Cytochrome cd1 nitrite reductases are periplasmic proteins involved in the second step of the denitrification pathway (NO3-!NO2-!NO!N2O!N2), corresponding to the reduction of nitrite to nitric oxide [1,2,3]. cd1NiRs are homodimeric proteins containing one ctype and one d1-type heme per subunit
The proteins were adsorbed on the self-assembled monolayers (SAMs)-coated electrodes following two different procedures: the modified electrode was i) immersed for 1 hour in a 0.1 μM protein solution, removed and rinsed with supporting electrolyte to eliminate unbound or loosely bound protein or ii) directly placed into the SERR spectroelectrochemical cell containing the supporting electrolyte and 0.1 μM protein, which was allowed to adsorb at open circuit for 30 min; positive or negative potentials were applied to the electrode during “in-cell” adsorption
We have focused on the formation and characterization of a functional catalytic complex between Mhcd1 and cyt c552

Summary

Introduction

Cytochrome cd1 nitrite reductases (cd1NiRs) are periplasmic proteins involved in the second step of the denitrification pathway (NO3-!NO2-!NO!N2O!N2), corresponding to the reduction of nitrite to nitric oxide [1,2,3]. cd1NiRs are homodimeric proteins containing one ctype and one d1-type heme per subunit. Successful co-immobilization of the two proteins on the same electrode construct was demonstrated by SERR (Fig 1), based on individual spectroscopic fingerprints of the ferric and ferrous cyt c552 and Mhcd1, which could be identified in the SERR spectra of the complex.

Results

Conclusion