Catalytic Importance of Acidic Amino Acids on Subunit NuoB of the Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I)

Dirk Flemming,Petra Hellwig,Simone Lepper,Daniel P Kloer,Thorsten Friedrich

doi:10.1074/jbc.m602499200

Dirk Flemming, Petra Hellwig + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m602499200

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Abstract

The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli is composed of 13 subunits called NuoA through NuoN and contains one FMN and 9 iron-sulfur clusters as redox groups. Electron transfer from NADH to ubiquinone is coupled with the translocation of protons across the membrane by a yet unknown mechanism. Redox-induced Fourier transform infrared difference spectroscopy showed that the oxidation of iron-sulfur cluster N2 located on NuoB is accompanied by the protonation of acidic amino acid(s). Here, we describe the effect of mutating the conserved acidic amino acids on NuoB. The complex was assembled in all mutants but the electron transfer activity was completely abolished in the mutants E67Q, D77N, and D94N. The complex isolated from these mutants contained N2 although in diminished amounts. The protonation of acidic amino acid(s) coupled with the oxidation of N2 was not detectable in the complex from the mutant E67Q. However, the conservative mutations E67D and D77E did not disturb the enzymatic activity, and the signals because of the protonation of acidic amino acid(s) were detectable in the E67D mutant. We discuss the possible participation of Glu(67) in a proton pathway coupled with the redox reaction of N2.

Highlights

The proton-translocating NADH:ubiquinone oxidoreductase (EC 1.6.99.3), called respiratory complex I,3 is the largest and least understood of the complexes of the respiratory chains [1,2,3,4,5]
If N2 participates in proton pumping, its redox reaction is coupled to a protonation/deprotonation of the cluster itself and/or of surrounding amino acids
We demonstrated by means of electrochemically induced FT-IR difference spectroscopy of complex I from site-directed mutants that the reduction of N2 is accompanied by the protonation of Tyr114 and Tyr139 on NuoB

Summary

EXPERIMENTAL PROCEDURES

Materials and Strains—E. coli strain ANN023 [19] is a derivative of strain AN387 [29] and was generated using a genomic replacement method as described [27]. The resulting plasmids containing the desired mutation were used for transformation of the deletion strain ANN023 In this strain, expression of NuoB and complementation of the chromosomal deletion was carried out by adding arabinose to a final concentration of 0.2% (w/v) as described [19]. The protein was equilibrated with an initial potential at the electrode, and single beam spectra in the vis and IR range were recorded. Catalytic Activity of the Mutant Strains—The E. coli strain ferricyanide reductase activity, respectively, to discriminate the ANN023 lacks subunit NuoB and is not able to assem- two membrane-bound NADH dehydrogenases of E. coli Strain ANN023 complemented with wild-type nuoB on complex I in the cytoplasmic membranes of the mutants can be estimated from the d-NADH/ferricyanide reductase assay (Table 1). The control strain ANN023/pBAD did not show any complex I activity

Complex I content was reduced by approximately one third in mutants

Sensitivity to piericidin A ϩϪϪϪϪϩϩϩϩϩϩϩϩ

Findings

DISCUSSION