Cecilia Hägerhäll

Transmembrane topology of the NuoL, M and N subunits of NADH:quinone oxidoreductase and their homologues among membrane-bound hydrogenases and bona fide antiporters

Nicotinamide adenine dinucleotide-reduced form (NADH):quinone oxidoreductase (respiratory Complex I), F420H2 oxidoreductase and complex, membrane-bound NiFe-hydrogenase contain protein subunits homologous to a certain type of bona fide antiporters. In Complex I, these polypeptides (NuoL/ND5, NuoM/ND4, NuoN/ND2) are most likely core components of the proton pumping mechanism, and it is thus important to learn more about their structure and function. In this work, we have determined the transmembrane topology of one such polypeptide, and built a 2D structural model of the protein valid for all the homologous polypeptides. The experimentally determined transmembrane topology was different from that predicted by majority vote hydrophobicity analyses of members of the superfamily. A detailed phylogenetic analysis of a large set of primary sequences shed light on the functional relatedness of these polypeptides.

A structural moDAl for the membrane-integral domain of succinate:quinone oxidoreductases

Many succinate:quinone oxidoreductases in bacteria and mitochondria, i.e. succinate:quinone reductases and fumarate reductases, contain in the membrane anchor a cytochrome b whose structure and function is poorly understood. Based on biochemical data and polypeptiDA sequence information, we show that the anchors in different organisms are related DAspite an apparent diversity in polypeptiDA and heme composition. A general structural moDAl for the membrane‐integral domain of the anchors is proposed. It is an antiparallel four‐helix bundle with a novel arrangement of hexa‐coordinated protoheme IX. The structure can be applied to a larger group of membrane‐integral cytochromes of b‐type and has evolutionary and functional implications.

The ‘antiporter module’ of respiratory chain Complex I includes the MrpC/NuoK subunit – a revision of the modular evolution scheme

Respiratory chain Complex I or NADH:quinone oxidoreductase catalyzes oxidation of NADH in the mitochondrial matrix or bacterial cytoplasm and reduction of quinone in the membrane, coupled to pumping of 4H+/2e− across the membrane. The same enzyme complex is also capable of the reverse reaction, i.e. Δμ H+ ‐supported NAD+ reduction. The molecular mechanism that couples electron transfer to proton pumping is not understood. The Complex I enzyme, containing 14 protein subunits necessary for function, has evolved from smaller functional building blocks. Three Complex I protein subunits, NuoL, NuoM and NuoN, show primary sequence similarity to one particular class of antiporters, and are thus predicted to play a role in the proton translocation machinery. These antiporters, MrpA and MrpD are encoded by a conserved gene cluster, that contains seven genes. In previous work we have determined that these antiporters come in two subclasses, MrpA‐type and MrpD‐type, and that the Complex I subunit NuoL is more closely related to MrpA and NuoM and N are more closely related to the MrpD antiporter. This implied that both MrpA and MrpD had been recruited to Complex I, rather than arising from gene duplications of one antiporter encoding gene. In this work we show that MrpC and NuoK are homologous proteins. The most plausible explanation for these findings is that a multisubunit antiporter complex was recruited to the ancestral enzyme. We further conclude that the last common ancestor of the Complex I enzyme family and membrane bound NiFe hydrogenases of type 3 and 4 contained the NuoKLMN subunit module.

EPR Characterization of Ubisemiquinones and Iron-Sulfur Cluster N2, Central Components of the Energy Coupling in the NADH-Ubiquinone Oxidoreductase (Complex I) In Situ

AbstractThe proton-translocating NADH-ubiquinone oxidoreductase (complex I) is the largest and least understood respiratory complex. The intrinsic redox components (FMN and iron–sulfur clusters) reside in the promontory part of the complex. Ubiquinone is the most possible key player in proton-pumping reactions in the membrane part. Here we report the presence of three distinct semiquinone species in complex I in situ, showing widely different spin relaxation profiles. As our first approach, the semiquinone forms were trapped during the steady state NADH-ubiquinone-1 (Q1) reactions in the tightly coupled, activated bovine heart submitochondrial particles, and were named SQNf (fast-relaxing component), SQNs (slow-relaxing), and SQNx (very slow relaxing). This indicates the presence of at least three different quinone-binding sites in complex I. In the current study, special attention was placed on the SQNf, because of its high sensitivities to

The Evolution of Respiratory Chain Complex I from a Smaller Last Common Ancestor Consisting of 11 Protein Subunits

\Delta \tilde \mu _{H^ + }

Transmembrane orientation and topology of the NADH:quinone oxidoreductase putative quinone binding subunit NuoH

and to specific complex I inhibitors (rotenone and piericidin A) in a unique manner. Rotenone inhibits the forward electron transfer reaction more strongly than the reverse reaction, while piericidine A inhibits both reactions with a similar potency. Rotenone quenched the SQNf signal at a much lower concentration than that required to quench the slower relaxing components (SQNs and SQNx). A close correlation was shown between the line shape alteration of the g‖ = 2.05 signal of the cluster N2 and the quenching of the SQNf signal, using two different experimental approaches: (1) changing the

Homologous protein subunits from Escherichia coli NADH:quinone oxidoreductase can functionally replace MrpA and MrpD in Bacillus subtilis

\Delta \tilde \mu _{H^ + }