Jürgen Koch

Transport of nuclear DNA into the cytoplasm in cultured animal cells. A survey

Recently several observations of a special class of cytoplasmic DNA in animal cells have been reported [l-5]. Apparently this DNA is replicated in the nucleus and is later transported to the cytoplasm. Results obtained with cultured human liver cells seem to rule out the possibility that this DNA stems from any random contamination of the cytoplasmic fractions with nuclear DNA during cell fractionation [ 11. So far a defined biological role could not be assigned to this DNA. However, the assumption seems reasonable that this DNA has a function in the transfer of information from the nucleus to the cytoplasm in the eukaryotic cell [3]. The importance of this notion prompted us to survey cells of several different species in order to see whether the phenomenon of the transport of DNA from the nucleus to the cytoplasm is a general one in animal cells.

Methanogenic heterodisulfide reductase (HdrABC-MvhAGD) uses two noncubane [4Fe-4S] clusters for reduction

Methanogenic archaea metabolism Most of the methane on Earth is produced by the metabolism of methanogenic archaea. The final step involves a reaction between methyl-coenzyme M and coenzyme B to give CoM-S-S-CoB and methane. Wagner et al. report a high-resolution structure of the methanogenic heterodisulfide reductase (HdtABC)-[NiFe]-hydrogenase, the enzyme that reduces the disulfide and couples this to the reduction of ferredoxin in an energy-conserving process known as flavin-based electron bifurcation (FBEB) (see the Perspective by Dobbek). The reduced ferredoxin, in turn, drives the first step of methanogenesis. The structure shows how two noncubane [4Fe-4S] clusters perform disulfide cleavage and gives insight into the mechanism of FBEB. Science, this issue p. 699; see also p. 642 Crystal structures reveal that two unconventional Fe-S clusters perform the challenging heterodisulfide reductase reaction. In methanogenic archaea, the carbon dioxide (CO2) fixation and methane-forming steps are linked through the heterodisulfide reductase (HdrABC)–[NiFe]-hydrogenase (MvhAGD) complex that uses flavin-based electron bifurcation to reduce ferredoxin and the heterodisulfide of coenzymes M and B. Here, we present the structure of the native heterododecameric HdrABC-MvhAGD complex at 2.15-angstrom resolution. HdrB contains two noncubane [4Fe-4S] clusters composed of fused [3Fe-4S]-[2Fe-2S] units sharing 1 iron (Fe) and 1 sulfur (S), which were coordinated at the CCG motifs. Soaking experiments showed that the heterodisulfide is clamped between the two noncubane [4Fe-4S] clusters and homolytically cleaved, forming coenzyme M and B bound to each iron. Coenzymes are consecutively released upon one-by-one electron transfer. The HdrABC-MvhAGD atomic model serves as a structural template for numerous HdrABC homologs involved in diverse microbial metabolic pathways.

Assignment of the [4Fe-4S] clusters of Ech hydrogenase from Methanosarcina barkeri to individual subunits via the characterization of site-directed mutants

Ech hydrogenase from Methanosarcina barkeri is a member of a distinct group of membrane‐bound [NiFe] hydrogenases with sequence similarity to energy‐conserving NADH:quinone oxidoreductase (complex I). The sequence of the enzyme predicts the binding of three [4Fe‐4S] clusters, one by subunit EchC and two by subunit EchF. Previous studies had shown that two of these clusters could be fully reduced under 105 Pa of H2 at pH 7 giving rise to two distinct S½ electron paramagnetic resonance (EPR) signals, designated as the g = 1.89 and the g = 1.92 signal. Redox titrations at different pH values demonstrated that these two clusters had a pH‐dependent midpoint potential indicating a function in ion pumping. To assign these signals to the subunits of the enzyme a set of M. barkeri mutants was generated in which seven of eight conserved cysteine residues in EchF were individually replaced by serine. EPR spectra recorded from the isolated mutant enzymes revealed a strong reduction or complete loss of the g = 1.92 signal whereas the g = 1.89 signal was still detectable as the major EPR signal in five mutant enzymes. It is concluded that the cluster giving rise to the g = 1.89 signal is the proximal cluster located in EchC and that the g = 1.92 signal results from one of the clusters of subunit EchF. The pH‐dependence of these two [4Fe‐4S] clusters suggests that they simultaneously mediate electron and proton transfer and thus could be an essential part of the proton‐translocating machinery.

H-matrix methods for linear and quasi-linear integral operators appearing in population balances

Abstract In population dynamics, the source term of breakage and the sink term of coagulation are often described by linear and quasi-linear operators, respectively. These operators are characterised by their kernel functions. Naive discretisation leads to full matrices and therefore to quadratic complexity O ( n 2 ) , if n denotes the number of degrees of freedom. For several popular kernel functions, e.g. the modified Smoluchowski kernel, an efficient treatment is introduced using the ideas of H -matrices. This approach leads to linear complexity O ( n ) .

Direct interaction of coenzyme M with the active-site Fe-S cluster of heterodisulfide reductase

Heterodisulfide reductase (HDR) catalyzes the formation of coenzyme M (CoM–SH) and coenzyme B (CoB–SH) by the reversible reduction of the heterodisulfide, CoM–S–S–CoB. This reaction recycles the two thiol coenzymes involved in the final step of microbial methanogenesis. Electron paramagnetic resonance (EPR) and variable‐temperature magnetic circular dichroism spectroscopic experiments on oxidized HDR incubated with CoM–SH revealed a S = 1/2 [4Fe–4S]3+ cluster, the EPR spectrum of which is broadened in the presence of CoM–33SH [Duin, E.C., Madadi‐Kahkesh, S., Hedderich, R., Clay, M.D. and Johnson, M.K. (2002) Heterodisulfide reductase from Methanothermobacter marburgensis contains an active‐site [4Fe–4S] cluster that is directly involved in mediating heterodisulfide reduction. FEBS Lett. 512, 263–268; Duin, E.C., Bauer, C., Jaun, B. and Hedderich, R. (2003) Coenzyme M binds to a [4Fe–4S] cluster in the active site of heterodisulfide reductase as deduced from EPR studies with the [33S]coenzyme M‐treated enzyme. FEBS Lett. 538, 81–84]. These results provide indirect evidence that the disulfide binds to the iron–sulfur cluster during reduction. We report here direct structural evidence for this interaction from Se X‐ray absorption spectroscopic investigation of HDR treated with the selenium analog of coenzyme M (CoM–SeH). Se K edge extended X‐ray absorption fine structure confirms a direct interaction of the Se in CoM–SeH‐treated HDR with an Fe atom of the Fe–S cluster at an Fe–Se distance of 2.4 Å.

H-matrix methods for quadratic integral operators appearing in population balances

Abstract In population dynamics the source term of breakage as well as the source and the sink term of coagulation are often described by an integral operator. All these operators are characterised by their kernel functions. Naive discretisation leads to full matrices and therefore to quadratic complexity O ( n 2 ) , if n denotes the number of degrees of freedom. In a recent paper [Koch, J., Hackbusch, W., & Sundmacher, K. (2007). H -matrix methods for linear and quasi-linear integral operators appearing in population balances. Computers & Chemical Engineering, 31 (7), 745–759] we have shown that for the source term of breakage and the sink term of coagulation, which are presented by linear and quasi-linear operators, it is possible to achieve a discretisation complexity of O ( n ) . For these two operators for further popular kernel functions the ideas of H -matrices were used. For the source term of coagulation, which is a quadratic operator, a more ambitious approach is necessary. We introduce a numerical treatment for two popular kernel functions combining the ideas of H -matrices and the fast Fourier transformation (FFT). This leads to almost linear complexity of O ( n log ⁡ n ) and O ( n log ⁡ 2 n ) but it turns unfortunately out that the new method is only applicable in a reasonable way for one of the two kernel functions.

Transport of nuclear DNA into the cytoplasm in animal cells The complexity of the transported DNA

The concept that the genetic information flows from DNA to RNA by means of a transcription process before it is expressed as protein (translation) stems from experiments with prokaryotic organisms [ 1, 21. In these organisms transcription and translation proceed in one spatial unity, a ternary association of DNA, RNA and ribosomes [3]. This concept has also been adopted for eukaryotic cells with the modification that a messenger RNA (mRNA) is synthesized in the nucleus and subsequently migrates to the cytoplasm where translation occurs. In other words transcription and translation are spatially separated by the nuclear membrane. However, the fraction of rapidly labeled heterogeneous RNA in the nucleus of animal cells that behaves as a precursor of cytoplasmic polyribosomal messenger RNA is quite small and the results of the experiments intended to demonstrate this migration of mRNA across the nuclear membrane were not altogether equivocal [4-71. More recently a special class of cytoplasmic DNA has been observed in chick cells [X, 91 and in human cells [9-121, the origin of which apparently is the cell nucleus [ 10, 131. Bell [ 141 already has hypothesized that this cytoplasmic DNA which he has called informational DNA (I-DNA) represents a fraction of the total genome and acts as a template for the synthesis of mRNA in the cytoplasm. In view of the experiments which indicate that this DNA is replicated within the nucleus and is later transported to the cytoplasm [ 10, 131 an alternative model for the information transfer across the nuclear membrane can be

Simulation of the population balance for droplet breakage in a liquid-liquid stirred tank reactor using H-matrix methods

Abstract In population balance equations particle breakage is often described by a Volterra integral operator. Naive discretisation of this operator leads to quadratic complexity. In this paper an efficient numerical treatment for Galerkin discretisation of the integral operator is suggested, based on the idea of H-matrices, which leads to linear complexity.