Correlating kinetic and structural data on ubiquinone binding and reduction by respiratory complex I

Respiratory complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in mammalian cells, powers ATP synthesis by using the energy from electron transfer from NADH to ubiquinone-10 to drive protons across the energy-transducing mitochondrial inner membrane. Ubiquinone-10 is extremely hydrophobic, but in complex I the binding site for its redox-active quinone headgroup is ~20 Å above the membrane surface. Structural data suggest it accesses the site by a narrow channel long enough to accommodate almost all its ~50 Å isoprenoid chain. However, how ubiquinone/ol exchange occurs on catalytically-relevant timescales, and whether binding/dissociation events are involved in coupling electron transfer to proton translocation, are unknown. Here, we use proteoliposomes containing complex I, together with a quinol oxidase, to determine the kinetics of complex I catalysis with ubiquinones of varying isoprenoid chain length, from 1 to 10 units. We interpret our results using structural data, which show the hydrophobic channel is interrupted by a highly-charged region at isoprenoids 4 to 7. We demonstrate that ubiquinol-10 dissociation is not rate determining, and deduce that ubiquinone-10 has both the highest binding affinity and the fastest binding rate. We propose that the charged region and chain directionality assist product dissociation, and that isoprenoid stepping ensures short transit times. These properties of the channel do not benefit the exhange of short-chain quinones, for which product dissociation may become rate limiting. Thus, we discuss how the long channel does not hinder catalysis under physiological conditions, and the possible roles of ubiquinone/ubiquinol binding/dissociation in energy conversion.We thank C. Humphreys & Sons Abattoir (Chelmsford) for providing bovine hearts and Sotiria Tavoulari for help with the amido black assay. Computing time was provided by the SuperMuc at The Leibniz Rechenzentrum (Grant pr48de). This work was supported by the Medical Research Council (Grant U105663141 to J.H.) and by the German Research Foundation (V.R.I.K.)

Late Replication Domains in Polytene and Non-Polytene Cells of Drosophila melanogaster

In D. melanogaster polytene chromosomes, intercalary heterochromatin (IH) appears as large dense bands scattered in euchromatin and comprises clusters of repressed genes. IH displays distinctly low gene density, indicative of their particular regulation. Genes embedded in IH replicate late in the S phase and become underreplicated. We asked whether localization and organization of these late-replicating domains is conserved in a distinct cell type. Using published comprehensive genome-wide chromatin annotation datasets (modENCODE and others), we compared IH organization in salivary gland cells and in a Kc cell line. We first established the borders of 60 IH regions on a molecular map, these regions containing underreplicated material and encompassing ∼12% of Drosophila genome. We showed that in Kc cells repressed chromatin constituted 97% of the sequences that corresponded to IH bands. This chromatin is depleted for ORC-2 binding and largely replicates late. Differences in replication timing between the cell types analyzed are local and affect only sub-regions but never whole IH bands. As a rule such differentially replicating sub-regions display open chromatin organization, which apparently results from cell-type specific gene expression of underlying genes. We conclude that repressed chromatin organization of IH is generally conserved in polytene and non-polytene cells. Yet, IH domains do not function as transcription- and replication-regulatory units, because differences in transcription and replication between cell types are not domain-wide, rather they are restricted to small “islands” embedded in these domains. IH regions can thus be defined as a special class of domains with low gene density, which have narrow temporal expression patterns, and so displaying relatively conserved organization

Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.

Complex I (NADH:ubiquinone oxidoreductase) uses the reducing potential of NADH to drive protons across the energy-transducing inner membrane and power oxidative phosphorylation in mammalian mitochondria. Recent cryo-EM analyses have produced near-complete models of all 45 subunits in the bovine, ovine and porcine complexes and have identified two states relevant to complex I in ischemia-reperfusion injury. Here, we describe the 3.3-Å structure of complex I from mouse heart mitochondria, a biomedically relevant model system, in the 'active' state. We reveal a nucleotide bound in subunit NDUFA10, a nucleoside kinase homolog, and define mechanistically critical elements in the mammalian enzyme. By comparisons with a 3.9-Å structure of the 'deactive' state and with known bacterial structures, we identify differences in helical geometry in the membrane domain that occur upon activation or that alter the positions of catalytically important charged residues. Our results demonstrate the capability of cryo-EM analyses to challenge and develop mechanistic models for mammalian complex I

Divalent Metal Ions Tune the Self-Splicing Reaction of the Yeast Mitochondrial Group II Intron Sc.ai5γ

Group II introns are large ribozymes, consisting of six functionally distinct domains that assemble in the presence of Mg2+ to the active structure catalyzing a variety of reactions. The first step of intron splicing is well characterized by a Michaelis–Menten-type cleavage reaction using a two-piece group II intron: the substrate RNA, the 5′-exon covalently linked to domains 1, 2, and 3, is cleaved upon addition of domain 5 acting as a catalyst. Here we investigate the effect of Ca2+, Mn2+, Ni2+, Zn2+, Cd2+, Pb2+, and [Co(NH3)6]3+ on the first step of splicing of the Saccharomyces cerevisiae mitochondrial group II intron Sc.ai5γ. We find that this group II intron is very sensitive to the presence of divalent metal ions other than Mg2+. For example, the presence of only 5% Ca2+ relative to Mg2+ results in a decrease in the maximal turnover rate k cat by 50%. Ca2+ thereby has a twofold effect: this metal ion interferes initially with folding, but then also competes directly with Mg2+ in the folded state, the latter being indicative of at least one specific Ca2+ binding pocket interfering directly with catalysis. Similar results are obtained with Mn2+, Cd2+, and [Co(NH3)6]3+. Ni2+ is a much more powerful inhibitor and the presence of either Zn2+ or Pb2+ leads to rapid degradation of the RNA. These results show a surprising sensitivity of such a large multidomain RNA on trace amounts of cations other than Mg2+ and raises the question of biological relevance at least in the case of Ca2+

Correlating kinetic and structural data on ubiquinone binding and reduction by respiratory complex I

Respiratory complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in mammalian cells, powers ATP synthesis by using the energy from electron transfer from NADH to ubiquinone-10 to drive protons across the energy-transducing mitochondrial inner membrane. Ubiquinone-10 is extremely hydrophobic, but in complex I the binding site for its redox-active quinone headgroup is ~20 Å above the membrane surface. Structural data suggest it accesses the site by a narrow channel long enough to accommodate almost all its ~50 Å isoprenoid chain. However, how ubiquinone/ol exchange occurs on catalytically-relevant timescales, and whether binding/dissociation events are involved in coupling electron transfer to proton translocation, are unknown. Here, we use proteoliposomes containing complex I, together with a quinol oxidase, to determine the kinetics of complex I catalysis with ubiquinones of varying isoprenoid chain length, from 1 to 10 units. We interpret our results using structural data, which show the hydrophobic channel is interrupted by a highly-charged region at isoprenoids 4 to 7. We demonstrate that ubiquinol-10 dissociation is not rate determining, and deduce that ubiquinone-10 has both the highest binding affinity and the fastest binding rate. We propose that the charged region and chain directionality assist product dissociation, and that isoprenoid stepping ensures short transit times. These properties of the channel do not benefit the exhange of short-chain quinones, for which product dissociation may become rate limiting. Thus, we discuss how the long channel does not hinder catalysis under physiological conditions, and the possible roles of ubiquinone/ubiquinol binding/dissociation in energy conversion.We thank C. Humphreys & Sons Abattoir (Chelmsford) for providing bovine hearts and Sotiria Tavoulari for help with the amido black assay. Computing time was provided by the SuperMuc at The Leibniz Rechenzentrum (Grant pr48de). This work was supported by the Medical Research Council (Grant U105663141 to J.H.) and by the German Research Foundation (V.R.I.K.)

Bathymetry Estimates in the Southern Oceans from SEASAT Altimetry

A mean sea surface height based on Seasat altimeter data has been high-pass filtered to reveal a pattern of geoid anomalies which are highly correlated with sea-floor topography. The technique is used to locate new bathymetric features in the poorly surveyed southern oceans