Biomedical subjects

Leonid A Sazanov

Publications and source records attributed to Leonid A Sazanov.

7 recordsLinked to original sources

PubMed2006

Projection structure of the membrane domain of Escherichia coli respiratory complex I at 8 A resolution.

Respiratory complex I (NADH:ubiquinone oxidoreductase) is an L-shaped multisubunit protein assembly consisting of a hydrophobic membrane arm and a hydrophilic peripheral arm. It catalyses the transfer of two electrons from NADH to quinone coupled to the translocation of four protons across the membrane. Although we have solved recently the crystal structure of the peripheral arm, the structure of the complete enzyme and the coupling mechanism are not yet known. The membrane domain of Escherichia coli complex I consists of seven different subunits with total molecular mass of 258 kDa. It is significantly more stable than the whole enzyme, which allowed us to obtain well-ordered two-dimensional crystals of the domain, belonging to the space group p22(1)2(1). Comparison of the projection map of negatively stained crystals with previously published low-resolution structures indicated that the characteristic curved shape of the membrane domain is remarkably well conserved between bacterial and mitochondrial enzymes, helping us to interpret projection maps in the context of the intact complex. Two pronounced stain-excluding densities at the distal end of the membrane domain are likely to represent the two large antiporter-like subunits NuoL and NuoM. Cryo-electron microscopy on frozen-hydrated crystals allowed us to calculate a projection map at 8 A resolution. About 60 transmembrane alpha-helices, both perpendicular to the membrane plane and tilted, are present within one membrane domain, which is consistent with secondary structure predictions. A possible binding site and access channel for quinone are found at the interface with the peripheral arm. Tentative assignment of individual subunits to the features of the map has been made. The location of subunits NuoL and NuoM at substantial distance from the peripheral arm, which contains all the redox centres of the complex, indicates that conformational changes are likely to play a role in the mechanism of coupling between electron transfer and proton pumping.

Cell Membrane↗

PubMed2006

Identification of a novel subunit of respiratory complex I from Thermus thermophilus.

The hydrophilic domain (peripheral arm) of the proton-translocating NADH:quinone oxidoreductase (complex I) from the thermophilic organism Thermus thermophilus HB8 has been purified and characterized. The subcomplex is stable in sodium dodecyl sulfate up to 80 degrees C. Of nine iron-sulfur clusters, four to five (one or two binuclear and three tetranuclear) could be detected by EPR in the NADH-reduced enzyme. The preparation consists of eight different polypeptides. Seven of them have been positively identified by peptide mass mapping and N-terminal sequencing as known hydrophilic subunits of T. thermophilus complex I. The eighth polypeptide copurified with the subcomplex at all stages, is strongly associated with the other subunits, and is present in crystals of the subcomplex, used for X-ray data collection. Therefore, it has been identified as a novel complex I subunit and named Nqo15. It is encoded in a locus separate from the nqo operon, containing the 14 other known complex I genes. ORFs encoding Nqo15 homologues are present in the genomes of the closest relatives of T. thermophilus. Our data show that, contrary to previous assumptions, bacterial complex I can contain proteins in addition to a "core" complement of 14 subunits.

Amino Acid Sequence↗

PubMed2006

Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus.

Respiratory complex I plays a central role in cellular energy production in bacteria and mitochondria. Its dysfunction is implicated in many human neurodegenerative diseases, as well as in aging. The crystal structure of the hydrophilic domain (peripheral arm) of complex I from Thermus thermophilus has been solved at 3.3 angstrom resolution. This subcomplex consists of eight subunits and contains all the redox centers of the enzyme, including nine iron-sulfur clusters. The primary electron acceptor, flavin-mononucleotide, is within electron transfer distance of cluster N3, leading to the main redox pathway, and of the distal cluster N1a, a possible antioxidant. The structure reveals new aspects of the mechanism and evolution of the enzyme. The terminal cluster N2 is coordinated, uniquely, by two consecutive cysteines. The novel subunit Nqo15 has a similar fold to the mitochondrial iron chaperone frataxin, and it may be involved in iron-sulfur cluster regeneration in the complex.

Animals↗

PubMed2005

Organization of iron-sulfur clusters in respiratory complex I.

Complex I of respiratory chains plays a central role in bioenergetics and is implicated in many human neurodegenerative diseases. An understanding of its mechanism requires a knowledge of the organization of redox centers. The arrangement of iron-sulfur clusters in the hydrophilic domain of complex I from Thermus thermophilus has been determined with the use of x-ray crystallography. One binuclear and six tetranuclear clusters are arranged, maximally 14 angstroms apart, in an 84-angstrom-long electron transfer chain. The binuclear cluster N1a and the tetranuclear cluster N7 are not in this pathway. Cluster N1a may play a role in the prevention of oxidative damage. The structure provides a framework for the interpretation of the large amounts of data accumulated on complex I.

Binding Sites↗

PubMed2004

Substrate-induced conformational change in bacterial complex I.

The mechanism coupling electron transfer and proton pumping in respiratory complex I (NADH-ubiquinone oxidoreductase) has not been established, but it has been suggested that it involves conformational changes. Here, the influence of substrates on the conformation of purified complex I from Escherichia coli was studied by cross-linking and electron microscopy. When a zero-length cross-linking reagent was used, the presence of NAD(P)H, in contrast to that of NAD+, prevented the formation of cross-links between the hydrophilic subunits of the complex, including NuoB, NuoI, and NuoCD. Comparisons using different cross-linkers suggested that NuoB, which is likely to coordinate the key iron-sulfur cluster N2, is the most mobile subunit. The presence of NAD(P)H led also to enhanced proteolysis of subunit NuoG. These data indicate that upon NAD(P)H binding, the peripheral arm of the complex adopts a more open conformation, with increased distances between subunits. Single particle analysis showed the nature of this conformational change. The enzyme retains its L-shape in the presence of NADH, but exhibits a significantly more open or expanded structure both in the peripheral arm and, unexpectedly, in the membrane domain also.

Electron Transport Complex I↗

PubMed2003

The location of NuoL and NuoM subunits in the membrane domain of the Escherichia coli complex I: implications for the mechanism of proton pumping.

The molecular organization of bacterial NADH: ubiquinone oxidoreductase (complex I or NDH-1) is not established, apart from a rough separation into dehydrogenase, connecting and membrane domains. In this work, complex I was purified from Escherichia coli and fragmented by replacing dodecylmaltoside with other detergents. Exchange into decyl maltoside led to the removal of the hydrophobic subunit NuoL from the otherwise intact complex. Diheptanoyl phosphocholine led to the loss of NuoL and NuoM subunits, whereas other subunits remained in the complex. The presence of N,N-dimethyldodecylamine N-oxide or Triton X-100 led to further disruption of the membrane domain into fragments containing NuoL/M/N, NuoA/K/N, and NuoH/J subunits. Among the hydrophilic subunits, NuoCD was most readily dissociated from the complex, whereas NuoB was partially dissociated from the peripheral arm assembly in N,N-dimethyldodecylamine N-oxide. A model of subunit arrangement in bacterial complex I based on these data is proposed. Subunits NuoL and NuoM, which are homologous to antiporters and are implicated in proton pumping, are located at the distal end of the membrane arm, spatially separated from the redox centers of the peripheral arm. This is consistent with proposals that the mechanism of proton pumping by complex I is likely to involve long range conformational changes.

Cell Membrane↗

PubMed2003

A role for native lipids in the stabilization and two-dimensional crystallization of the Escherichia coli NADH-ubiquinone oxidoreductase (complex I).

NADH-ubiquinone oxidoreductase (complex I or NDH-1) was purified from the BL21 strain of Escherichia coli using an improved procedure. The complex was effectively stabilized by addition of divalent cations and lipids, making the preparation suitable for structural studies. The ubiquinone reductase activity of the enzyme was fully restored by addition of native E. coli lipids. Two different two-dimensional crystal forms, with p2 and p3 symmetry, were obtained using lipids containing native E. coli extracts. Analysis of the crystals showed that they are formed by fully intact complex I in an L-shaped conformation. Activity assays and single particle analysis indicated that complex I maintains this structure in detergent solution and does not adopt a different conformation in the active state. Thus, we provide the first experimental evidence that complex I from E. coli has an L-shape in a lipid bilayer and confirm that this is also the case for the active enzyme in solution. This suggests strongly that bacterial complex I exists in an L-shaped conformation in vivo. Our results also indicate that native lipids play an important role in the activation, stabilization and, as a consequence, crystallization of purified complex I from E. coli.

Amino Acid Sequence↗