DOI: 10.1073/pnas.96.7.4149. NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron

NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron–sulfur cluster N2 to quinone

10.1073/pnas.96.7.4149

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

The proton-translocating NADH-quinone oxidoreductase (EC1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) inParacoccus denitrificansandThermus thermophilusHB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron–sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[3H]pyridaben ([3H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes ofP. denitrificansandT. thermophilusestablished photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron–sulfur cluster N2 to quinone.

Bibliography

Schuler, F., Yano, T., Di Bernardo, S., Yagi, T., Yankovskaya, V., Singer, T. P., & Casida, J. E. (1999). NADH-quinone oxidoreductase: PSST subunit couples electron transfer from ironâsulfur cluster N2 to quinone. Proceedings of the National Academy of Sciences, 96(7), 4149â4153.

Authors 7

Franz Schuler (first)
Takahiro Yano (additional)
Salvatore Di Bernardo (additional)
Takao Yagi (additional)
Victoria Yankovskaya (additional)
Thomas P. Singer (additional)
John E. Casida (additional)

References 37 Referenced 142

10.1017/S003358350000425X
Ernster L. Luft R. & Orrenius S. eds. (1995) Mitochondrial Diseases Nobel Symposium 90 Biochim. Biophys. Acta (Special Issue) 1271 1–292. (10.1016/0925-4439(95)90019-5)
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T P Singer Methods of Biochemical Analysis, ed D Glick (Wiley, New York) 22, 123–175 (1974). (10.1002/9780470110423.ch3) / Methods of Biochemical Analysis by Singer T P (1974)
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10.1021/bi00161a022

Dates

Type	When
Created	23 years, 1 month ago (July 26, 2002, 10:39 a.m.)
Deposited	1 year, 7 months ago (Jan. 4, 2024, midnight)
Indexed	2 weeks, 5 days ago (Aug. 7, 2025, 4:51 a.m.)
Issued	26 years, 4 months ago (March 30, 1999)
Published	26 years, 4 months ago (March 30, 1999)
Published Online	26 years, 4 months ago (March 30, 1999)
Published Print	26 years, 4 months ago (March 30, 1999)

Funders 0

None

BibTeX

@article{Schuler_1999, title={NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron–sulfur cluster N2 to quinone}, volume={96}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.96.7.4149}, DOI={10.1073/pnas.96.7.4149}, number={7}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Schuler, Franz and Yano, Takahiro and Di Bernardo, Salvatore and Yagi, Takao and Yankovskaya, Victoria and Singer, Thomas P. and Casida, John E.}, year={1999}, month=mar, pages={4149–4153} }

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