Electrostatic Interactions Between FeS Clusters in NADH:Ubiquinone Oxidoreductase (Complex I) from Escherichia coli

Abstract

The redox properties of the cofactors of NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli were studied by following the changes in electron paramagnetic resonance (EPR) and optical spectra upon electrochemical redox titration of the purified protein. At neutral pH, the FMN cofactor had a midpoint redox potential ( E m) approximately -350 mV ( n = 2). Binuclear FeS clusters were well-characterized: N1a was titrated with a single ( n = 1) transition, and E m = -235 mV. In contrast, the titration of N1b can only be fitted with the sum of at least two one-electron Nernstian curves with E m values of -245 and -320 mV. The tetranuclear clusters can also be separated into two groups, either having a single, n = 1, or more complex redox titration curves. The titration curves of the EPR bands attributed to the tetranuclear clusters N2 ( g = 2.045 and g = 1.895) and N6b ( g = 2.089 and g = 1.877) can be presented by the sum of at least two components, each with E m (app) approximately -200/-300 mV and -235/-315 mV, respectively. The titration of the signals at g = 1.956-1.947 (N3 or N7, E m = -315 mV), g = 2.022, and g = 1.932 (Nx, -365 mV) and the low temperature signal at g = 1.929 (N4 or N5, -330 mV) followed Nernstian n = 1 curves. The observed redox titration curves are discussed in terms of intrinsic electrostatic interactions between FeS centers in complex I. A model showing shifts of E m due to the electrostatic interaction between the centers is presented.

Structural Basis for the Mechanism of Respiratory Complex I

“…EPR studies of the intact complex suggest that although in the NADHreduced enzyme cluster N6b is likely to be reduced in addition to N2, N6a may not be (34,46,47). Therefore it is likely that the shift of the four-helix bundle, which occurs in this case (RND structure), is driven by the reduction of cluster N6b or N6b/N2 as a pair.…”

Reduction of the Iron−Sulfur Clusters in Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I) by EuII-DTPA, a Very Low Potential Reductant

“…There is now sufficient evidence to conclude that the spectrum observed in mitochondrial complex I is from N1b alone (10,13,26), so that the reduction potential of N1a is not in fact known, and N1b exhibits an extended potential dependence (25). In E. coli complex I N1a has a higher potential (from comparison of the cluster potentials in the overexpressed subunits (16)), and it is clearly visible in spectra recorded at around 40 K (10,12,27).…”

Electrostatics of the FeS clusters in respiratory complex I

“…Electron paramagnetic resonance (EPR) has been the most successful method of investigating the ET process, but due to the large number of redox centers and the overall complexity of the electron transport chain unambiguous interpretation of EPR experiments have been difficult [8, 9]. There has been significant controversy over the assignment of various spectral features to individual FeS clusters within the enzyme, and unambiguous assignments are still not available [9, 10]. Key to understanding ET in complex I is detailed knowledge of the midpoint reduction potentials (E m ) of the FeS clusters.…”

“…Key to understanding ET in complex I is detailed knowledge of the midpoint reduction potentials (E m ) of the FeS clusters. The binuclear N1a and tetranuclear N2 centers have unique redox potentials of −370 mV and −100 mV respectively, although a broad range of values have been reported for both clusters [4, 10]. The remaining clusters appear to be equipotential with E m values near −250 mV.…”

“…The remaining clusters appear to be equipotential with E m values near −250 mV. Recently Euro et al [10] showed that a number of the FeS clusters in complex I exhibit redox titration curves which are best modeled as the sum of two n=1 Nernstian curves indicating strong electrostatic interactions between redox centers. Furthermore, they introduced a thermodynamic model of the equilibrium redox state of the enzyme which includes explicit interactions between neighboring FeS clusters.…”