Using Hyperfine Electron Paramagnetic Resonance Spectroscopy to Define the Proton-Coupled Electron Transfer Reaction at Fe-S Cluster N2 in Respiratory Complex I.

Nolwenn Le Breton,Enrico Salvadori,Hannah R. Bridges,Andrew J. Y. Jones,Judy Hirst,Maxie M. Roessler,John J. Wright

doi:10.1021/jacs.7b09261

Abstract

Energy-transducing respiratory complex I (NADH:ubiquinone oxidoreductase) is one of the largest and most complicated enzymes in mammalian cells. Here, we used hyperfine electron paramagnetic resonance (EPR) spectroscopic methods, combined with site-directed mutagenesis, to determine the mechanism of a single proton-coupled electron transfer reaction at one of eight iron-sulfur clusters in complex I, [4Fe-4S] cluster N2. N2 is the terminal cluster of the enzyme's intramolecular electron-transfer chain and the electron donor to ubiquinone. Because of its position and pH-dependent reduction potential, N2 has long been considered a candidate for the elusive "energy-coupling" site in complex I at which energy generated by the redox reaction is used to initiate proton translocation. Here, we used hyperfine sublevel correlation (HYSCORE) spectroscopy, including relaxation-filtered hyperfine and single-matched resonance transfer (SMART) HYSCORE, to detect two weakly coupled exchangeable protons near N2. We assign the larger coupling with A(1H) = [-3.0, -3.0, 8.7] MHz to the exchangeable proton of a conserved histidine and conclude that the histidine is hydrogen-bonded to N2, tuning its reduction potential. The histidine protonation state responds to the cluster oxidation state, but the two are not coupled sufficiently strongly to catalyze a stoichiometric and efficient energy transduction reaction. We thus exclude cluster N2, despite its proton-coupled electron transfer chemistry, as the energy-coupling site in complex I. Our work demonstrates the capability of pulse EPR methods for providing detailed information on the properties of individual protons in even the most challenging of energy-converting enzymes.

Highlights

Mitochondrial complex I (NADH:ubiquinone oxidoreductase) is a redox-coupled proton pumping enzyme that is essential for respiration
To establish unambiguously the pH dependence of the reduction potential of N2 in mammalian complex I, we carried out a series of electron paramagnetic resonance (EPR)-monitored small-volume redox titrations, at pH values from 5 to 9, on the bovine enzyme (Supplementary Figure 1)
Because bovine complex I proved to be unstable at pH

Summary

■ INTRODUCTION

Mitochondrial complex I (NADH:ubiquinone oxidoreductase) is a redox-coupled proton pumping enzyme that is essential for respiration. We define the pHdependent reduction potential of N2 in the mammalian complex using small-volume potentiometric titrations[32] under tightly defined conditions: we develop a widely applicable buffer system over a wide pH range that accounts for temperature-dependent changes in pH during sample preparation that could otherwise lead to misleading results.[32,33] we apply pulse EPR measurements to detect exchangeable protons in the vicinity of the reduced N2 cluster. The [wild-type − H226M] difference spectrum (pH 6.5) clearly reveals a single set of proton cross-peaks (Figure 5A) with hyperfine coupling parameters that match H1 (Table 1). Neither the redox reaction or proton translocation is severely impaired by the absence of His190/226, and we conclude that proton-coupled electron transfer at cluster N2 is not required for complex I catalysis

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES