New insight into the mechanism of mitochondrial cytochrome c function.

We investigate functional role of the P76GTKMIFA83 fragment of the primary structure of cytochrome c. Based on the data obtained by the analysis of informational structure (ANIS), we propose a model of functioning of cytochrome c. According to this model, conformational rearrangements of the P76GTKMIFA83 loop fragment have a significant effect on conformational mobility of the heme. It is suggested that the conformational mobility of cytochrome c heme is responsible for its optimal orientation with respect to electron donor and acceptor within ubiquinol-cytochrome c oxidoreductase (complex III) and cytochrome c oxidase (complex IV), respectively, thus, ensuring electron transfer from complex III to complex IV. To validate the model, we design several mutant variants of horse cytochrome c with multiple substitutions of amino acid residues in the P76GTKMIFA83 sequence that reduce its ability to undergo conformational rearrangements. With this, we study the succinate-cytochrome c reductase and cytochrome c oxidase activities of rat liver mitoplasts in the presence of mutant variants of cytochrome c. The electron transport activity of the mutant variants decreases to different extent. Resonance Raman spectroscopy (RRS) and surface-enhanced Raman spectroscopy (SERS) data demonstrate, that all mutant cytochromes possess heme with the higher degree of ruffling deformation, than that of the wild-type (WT) cytochrome c. The increase in the ruffled deformation of the heme of oxidized cytochromes correlated with the decrease in the electron transport rate of ubiquinol-cytochrome c reductase (complex III). Besides, all mutant cytochromes have lower mobility of the pyrrol rings and methine bridges, than WT cytochrome c. We show that a decrease in electron transport activity in the mutant variants correlates with conformational changes and reduced mobility of heme porphyrin. This points to a significant role of the P76GTKMIFA83 fragment in the electron transport function of cytochrome c

Weichselian and Holocene palaeoenvironmental history of the Bol'shoy Lyakhovsky Island, New Siberian Archipelago, Arctic Siberia

Cryolithological, ground ice and fossil bioindicator (pollen, diatoms, plant macrofossils, rhizopods, insects, mammal bones) records from Bol'shoy Lyakhovsky Island permafrost sequences (73°20′N, 141°30′E) document the environmental history in the region for the past c. 115 kyr. Vegetation similar to modern subarctic tundra communities prevailed during the Eemian/Early Weichselian transition with a climate warmer than the present. Sparse tundra-like vegetation and harsher climate conditions were predominant during the Early Weichselian. The Middle Weichselian deposits contain peat and peaty soil horizons with bioindicators documenting climate amelioration. Although dwarf willows grew in more protected places, tundra and steppe vegetation prevailed. Climate conditions became colder and drier c. 30 kyr BP. No sediments dated between c. 28.5 and 12.05 14C kyr BP were found, which may reflect active erosion during that time. Herb and shrubby vegetation were predominant 11.6–11.3 14C kyr BP. Summer temperatures were c. 4 °C higher than today. Typical arctic environments prevailed around 10.5 14C kyr BP. Shrub alder and dwarf birch tundra were predominant between c. 9 and 7.6 kyr BP. Reconstructed summer temperatures were at least 4 °C higher than present. However, insect remains reflect that steppe-like habitats existed until c. 8 kyr BP. After 7.6 kyr BP, shrubs gradually disappeared and the vegetation cover became similar to that of modern tundra. Pollen and beetles indicate a severe arctic environment c. 3.7 kyr BP. However, Betula nana, absent on the island today, was still present. Together with our previous study on Bol'shoy Lyakhovsky Island covering the period between about 200 and 115 kyr, a comprehensive terrestrial palaeoenvironmental data set from this area in western Beringia is now available for the past two glacial–interglacial cycles

The intensity ratios of selected peaks in the RRS spectra of the reduced wild-type cytochrome <i>c</i> and its mutants.

<p>Data are shown as mean values ± SE. *p<0.05 compared to wild-type cytochrome <i>c</i> (non-parametric Kruskal-Wallis test with Dunn's multiple comparison test).</p

SERS study of oxidized cytochromes.

<p><b>(A)</b> The SERS spectra of the studied cytochromes in the oxidized state: WT—wild type; M1—T78N/K79Y/M80I/I81M/F82N, M2—T78S/K79P, M3—I81Y/A83Y/G84N. For clearer presentation, the spectra are shifted in vertical position. Numbers above peaks indicate positions of their maxima used in the analysis. X axis is a frequency shift, cm^-1; Y axis is SERS intensity, a.u. (B) Structural formula of heme <i>c</i> with numeration of C atoms.</p

Resonance Raman study of reduced cytochromes.

<p>The RRS spectra of the studied cytochromes in the reduced state: WT—wild type; M1—T78N/K79Y/M80I/I81M/F82N, M2—T78S/K79P, M3—I81Y/A83Y/G84N. For clearer presentation, the spectra are shifted in vertical position. X axis is a frequency shift, cm^-1 and Y axis is RRS intensity, a.u.</p

The informational structure of horse cytochrome <i>c</i> and its mutant forms: Result of the analyzis of the amino acid sequence using the ANIS method.

<p>(A) The hierarchically organized highest rank ELIS (continuous lines) and the fragments of the bipartite graph that cannot be revealed using the ANIS method (dashed line). X axis is the size of the smoothing interval a/2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178280#pone.0178280.ref021" target="_blank">21</a>], Y axis is the number N of amino acid in the primary structure of horse cytochrome <i>c</i>; (B), (C), (D) The hierarchically organized highest rank ELIS in mutant forms T78S/K79P, I81Y/A83Y/G84N, T78N/K79Y/M80I/I81M/F82N, respectively; (E) The spatial structure of horse cytochrome <i>c</i> (1HRC.PDB). The highest rank ELIS in the spatial structure of cytochrome c are shown. His18 and Met 80 residues coordinated to the Fe atom are indicated. The ADD- site (P76-A83) with the abnormally low density of first rank ELIS is shown by the arrows.</p

Comparison of the kinetic parameters of the succinate:cytochrome <i>c</i> reductase and cytochrome <i>c</i> oxidase reactions in rat liver mitoplasts in the presence of the externally added WT or mutant cytochromes <i>c</i>.

<p>The ratios of the maximum reaction rates in the presence of mutant forms of cytochrome <i>c</i> to the reaction rate in the presence of a WT protein <i>A</i>max(mut)/<i>A</i>max(wt) are also calculated.</p

The intensity ratios of selected peaks in the SERS spectra of oxidized wild-type cytochrome <i>c</i> and its mutants.

<p>Data are shown as mean values ± SE. *p<0.05 compared to wild-type cytochrome <i>c</i> (the non-parametric Kruskal-Wallis test with Dunn's multiple comparison test).</p

The structure of the oligonucleotides (direct primers) used to introduce mutations into a sequence of horse cytochrome <i>c</i>.

<p>The structure of the oligonucleotides (direct primers) used to introduce mutations into a sequence of horse cytochrome <i>c</i>.</p