S4.21 Monitoring the conformational change of the NADH dehydrogenase fragment from the E. coli complex I by attenuated total reflexion spectroscopy

ABSTRACT Complex I (EC 1.6.99.3 ) of the bacterium Escherichia coli is considered to be the minimal form of the type I NADH dehydrogenase, the first enzyme complex in the respiratory chain. Because of its small size and relative simplicity, the E. coli enzyme has become a model used to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex. To begin dissecting the processes by which E. coli cells regulate the expression ofnuo and the assembly of complex I, we undertook a genetic analysis of the nuo locus, which encodes the 14 Nuo subunits comprising E. coli complex I. Here we present the results of studies, performed on an isogenic collection ofnuo mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits. In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of nuo expression and/or the assembly of complex I.

Download Full-text

TAT-Conjugated NDUFS8 Can Be Transduced into Mitochondria in a Membrane-Potential-Independent Manner and Rescue Complex I Deficiency

International Journal of Molecular Sciences ◽

10.3390/ijms22126524 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6524

Author(s):

Bo-Yu Lin ◽

Gui-Teng Zheng ◽

Kai-Wen Teng ◽

Juan-Yu Chang ◽

Chao-Chang Lee ◽

...

Keyword(s):

Membrane Potential ◽

Fusion Proteins ◽

Complex I ◽

Nadh Dehydrogenase ◽

Leigh Syndrome ◽

Protein Transduction ◽

Independent Manner ◽

Complex I Deficiency ◽

Transactivator Of Transcription ◽

Hiv 1

NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8) is a nuclear-encoded core subunit of human mitochondrial complex I. Defects in NDUFS8 are associated with Leigh syndrome and encephalomyopathy. Cell-penetrating peptide derived from the HIV-1 transactivator of transcription protein (TAT) has been successfully applied as a carrier to bring fusion proteins into cells without compromising the biological function of the cargoes. In this study, we developed a TAT-mediated protein transduction system to rescue complex I deficiency caused by NDUFS8 defects. Two fusion proteins (TAT-NDUFS8 and NDUFS8-TAT) were exogenously expressed and purified from Escherichia coli for transduction of human cells. In addition, similar constructs were generated and used in transfection studies for comparison. The results showed that both exogenous TAT-NDUFS8 and NDUFS8-TAT were delivered into mitochondria and correctly processed. Interestingly, the mitochondrial import of TAT-containing NDUFS8 was independent of mitochondrial membrane potential. Treatment with TAT-NDUFS8 not only significantly improved the assembly of complex I in an NDUFS8-deficient cell line, but also partially rescued complex I functions both in the in-gel activity assay and the oxygen consumption assay. Our current findings suggest the considerable potential of applying the TAT-mediated protein transduction system for treatment of complex I deficiency.

Download Full-text

Molecular Gene Therapy: Overexpression of the Alternative NADH Dehydrogenase NDI1 Restores Overall Physiology in a Fungal Model of Respiratory Complex I Deficiency

Journal of Molecular Biology ◽

10.1016/j.jmb.2010.04.015 ◽

2010 ◽

Vol 399 (1) ◽

pp. 31-40 ◽

Cited By ~ 18

Author(s):

Marc F.P.M. Maas ◽

Carole H. Sellem ◽

Frank Krause ◽

Norbert A. Dencher ◽

Annie Sainsard-Chanet

Keyword(s):

Gene Therapy ◽

Complex I ◽

Nadh Dehydrogenase ◽

Respiratory Complex ◽

Complex I Deficiency ◽

Respiratory Complex I

Download Full-text

Cross‐linking study of membrane subunits K, L, M and N in E. coli complex I (997.10)

The FASEB Journal ◽

10.1096/fasebj.28.1_supplement.997.10 ◽

2014 ◽

Vol 28 (S1) ◽

Author(s):

Steven Vik ◽

Shaotong Zhu ◽

Ablat Tursun

Keyword(s):

Complex I ◽

Cross Linking ◽

E Coli

Download Full-text

Physiological relevance, localization and substrate specificity of the alternative (type II) mitochondrial NADH dehydrogenases of Ogataea parapolymorpha

10.1101/2021.04.28.441406 ◽

2021 ◽

Author(s):

Hannes Juergens ◽

Álvaro Mielgo-Gómez ◽

Albert Godoy-Hernández ◽

Jolanda ter Horst ◽

Janine M. Nijenhuis ◽

...

Keyword(s):

Substrate Specificity ◽

Respiratory Chain ◽

Complex I ◽

Nadh Dehydrogenase ◽

Physiological Role ◽

Proton Pumping ◽

Type Ii ◽

Respiratory Chains ◽

Alternative Type ◽

Nadh Dehydrogenases

AbstractMitochondria from Ogataea parapolymorpha harbor a branched electron-transport chain containing a proton-pumping Complex I NADH dehydrogenase and three alternative (type II) NADH dehydrogenases (NDH2s). To investigate the physiological role, localization and substrate specificity of these enzymes, growth of various NADH dehydrogenase mutants was quantitatively characterized in shake-flask and chemostat cultures, followed by oxygen-uptake experiments with isolated mitochondria. Furthermore, NAD(P)H:quinone oxidoreduction of the three NDH2s were individually assessed. Our findings show that the O. parapolymorpha respiratory chain contains an internal NADH-accepting NDH2 (Ndh2-1/OpNdi1), at least one external NAD(P)H-accepting enzyme and likely additional mechanisms for respiration-linked oxidation of cytosolic NADH. Metabolic regulation appears to prevent competition between OpNdi1 and Complex I for mitochondrial NADH. With the exception of OpNdi1, the respiratory chain of O. parapolymorpha exhibits metabolic redundancy and tolerates deletion of multiple NADH-dehydrogenase genes without compromising fully respiratory metabolism.ImportanceTo achieve high productivity and yields in microbial bioprocesses, efficient use of the energy substrate is essential. Organisms with branched respiratory chains can respire via the energy-efficient proton-pumping Complex I, or make use of alternative NADH dehydrogenases (NDH2s). The yeast Ogataea parapolymorpha contains three uncharacterized, putative NDH2s which were investigated in this work. We show that O. parapolymorpha contains at least one ‘internal’ NDH2, which provides an alternative to Complex I for mitochondrial NADH oxidation, albeit at a lower efficiency. The use of this NDH2 appeared to be limited to carbon excess conditions and the O. parapolymorpha respiratory chain tolerated multiple deletions without compromising respiratory metabolism, highlighting opportunities for metabolic (redox) engineering. By providing a more comprehensive understanding of the physiological role of NDH2s, including insights into their metabolic capacity, orientation and substrate specificity this study also extends our fundamental understanding of respiration in organisms with branched respiratory chains.

Download Full-text

The mitochondrial AAA protease FTSH3 regulates Complex I abundance by promoting its disassembly

Plant Physiology ◽

10.1093/plphys/kiab074 ◽

2021 ◽

Author(s):

Aneta Ivanova ◽

Abi S Ghifari ◽

Oliver Berkowitz ◽

James Whelan ◽

Monika W Murcha

Keyword(s):

Complex I ◽

Nadh Dehydrogenase ◽

Slow Growth ◽

Nadh:Ubiquinone Oxidoreductase ◽

Temperature Sensitive ◽

Wild Type ◽

Forward Genetic Screen ◽

Slow Growth Rate ◽

Partial Restoration ◽

Screen Approach

Abstract ATP is generated in mitochondria by oxidative phosphorylation. Complex I (NADH:ubiquinone oxidoreductase or NADH dehydrogenase) is the first multisubunit protein complex of this pathway, oxidising NADH and transferring electrons to the ubiquinone pool. Typically Complex I mutants display a slow growth rate compared to wild-type plants. Here, using a forward genetic screen approach for restored growth of a Complex I mutant, we have identified the mitochondrial ATP dependent metalloprotease, Filamentous Temperature Sensitive H 3 (FTSH3), as a factor that is required for the disassembly of Complex I. An ethyl methanesulfonate-induced mutation in FTSH3, named rmb1 (restoration of mitochondrial biogenesis 1), restored Complex I abundance and plant growth. Complementation could be achieved with FTSH3 lacking proteolytic activity, suggesting the unfoldase function of FTSH3 has a role in Complex I disassembly. The introduction of the rmb1 to an additional, independent, and extensively characterised Complex I mutant, ndufs4, resulted in similar increases to Complex I abundance and a partial restoration of growth. These results show that disassembly or degradation of Complex I plays a role in determining its steady-state abundance and thus turnover may vary under different conditions.

Download Full-text

Leishmania type II dehydrogenase is essential for parasite viability irrespective of the presence of an active complex I

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2103803118 ◽

2021 ◽

Vol 118 (42) ◽

pp. e2103803118

Author(s):

Margarida Duarte ◽

Cleide Ferreira ◽

Gurleen Kaur Khandpur ◽

Tamara Flohr ◽

Jannik Zimmermann ◽

...

Keyword(s):

Complex I ◽

Nadh Dehydrogenase ◽

Leishmania Major ◽

Proton Translocation ◽

Protozoan Parasite ◽

Type I ◽

Type Ii ◽

Active Complex ◽

Mitochondrial Inner Membrane ◽

Candidate Target

Type II NADH dehydrogenases (NDH2) are monotopic enzymes present in the external or internal face of the mitochondrial inner membrane that contribute to NADH/NAD+ balance by conveying electrons from NADH to ubiquinone without coupled proton translocation. Herein, we characterize the product of a gene present in all species of the human protozoan parasite Leishmania as a bona fide, matrix-oriented, type II NADH dehydrogenase. Within mitochondria, this respiratory activity concurs with that of type I NADH dehydrogenase (complex I) in some Leishmania species but not others. To query the significance of NDH2 in parasite physiology, we attempted its genetic disruption in two parasite species, exhibiting a silent (Leishmania infantum, Li) and a fully operational (Leishmania major, Lm) complex I. Strikingly, this analysis revealed that NDH2 abrogation is not tolerated by Leishmania, not even by complex I–expressing Lm species. Conversely, complex I is dispensable in both species, provided that NDH2 is sufficiently expressed. That a type II dehydrogenase is essential even in the presence of an active complex I places Leishmania NADH metabolism into an entirely unique perspective and suggests unexplored functions for NDH2 that span beyond its complex I–overlapping activities. Notably, by showing that the essential character of NDH2 extends to the disease-causing stage of Leishmania, we genetically validate NDH2—an enzyme without a counterpart in mammals—as a candidate target for leishmanicidal drugs.

Download Full-text