While a general model of H2 activation has been proposed for [FeFe]-hydrogenases, the structural and biophysical properties of the intermediates of the H-cluster catalytic site have not yet been discretely defined. Electron paramagnetic resonance (EPR) spectroscopy and Fourier transform infrared (FTIR) spectroscopy were used to characterize the H-cluster catalytic site, a [4Fe-4S]H subcluster linked by a cysteine thiolate to an organometallic diiron subsite with CO, CN, and dithiolate ligands, in [FeFe]-hydrogenase HydA1 from Chlamydomonas reinhardtii (CrHydA1). Oxidized CrHydA1 displayed a rhombic 2.1 EPR signal (g = 2.100, 2.039, 1.997) and an FTIR spectrum previously assigned to the oxidized H-cluster (Hox). Reduction of the Hox sample with 100% H2 or sodium dithionite (NaDT) nearly eliminated the 2.1 signal, which coincided with appearance of a broad 2.3-2.07 signal (g = 2.3-2.07, 1.863) and/or a rhombic 2.08 signal (g = 2.077, 1.935, 1.880). Both signals displayed relaxation properties similar to those of [4Fe-4S] clusters and are consistent with an S = 1/2 H-cluster containing a [4Fe-4S]H(+) subcluster. These EPR signals were correlated with differences in the CO and CN ligand modes in the FTIR spectra of H2- and NaDT-reduced samples compared with Hox. The results indicate that reduction of [4Fe-4S]H from the 2+ state to the 1+ state occurs during both catalytic H2 activation and proton reduction and is accompanied by structural rearrangements of the diiron subsite CO/CN ligand field. Changes in the [4Fe-4S]H oxidation state occur in electron exchange with the diiron subsite during catalysis and mediate electron transfer with either external carriers or accessory FeS clusters.
The role of Lys-63 ubiquitin chains in targeting proteins for proteasomal degradation is still obscure. We systematically compared proteasomal processing of Lys-63 ubiquitin chains with that of the canonical proteolytic signal, Lys-48 ubiquitin chains. Quantitative mass spectrometric analysis of ubiquitin chains in HeLa cells determines that the levels of Lys-63 ubiquitin chains are insensitive to short-time proteasome inhibition. Also, the Lys-48/Lys-63 ratio in the 26 S proteasome-bound fraction is 1.7-fold more than that in the cell lysates, likely because some cellular Lys-63 ubiquitin conjugates are sequestered by Lys-63 chain-specific binding proteins. In vitro, Lys-48 and Lys-63 ubiquitin chains bind the 26 S proteasome comparably, whereas Lys-63 chains are deubiquitinated 6-fold faster than Lys-48 chains. Also, Lys-63 tetraubiquitin-conjugated UbcH10 is rapidly deubiquitinated into the monoubiquitinated form, whereas Lys-48 tetraubiquitin targets UbcH10 for degradation. Furthermore, we found that both the ubiquitin aldehyde-and 1,10-phenanthroline-sensitive deubiquitinating activities of the 26 S proteasome contribute to Lys-48-and Lys-63-linkage deubiquitination, albeit the inhibitory extents are different. Together, our findings suggest that compared with Lys-48 chains, cellular Lys-63 chains have less proteasomal accessibility, and proteasome-bound Lys-63 chains are more rapidly deubiquitinated, which could cause inefficient degradation of Lys-63 conjugates.
In eukaryotic cells, cohesion between sister chromatids allows chromosomes to biorient on the metaphase plate and holds them together until they separate into daughter cells during mitosis. Cohesion is mediated by the cohesin protein complex. Although the association of this complex with particular regions of the genome is highly reproducible, it is unclear what distinguishes a chromosomal region for cohesin association. Since one of the primary locations of cohesin is intergenic regions between converging transcription units, we explored the relationship between transcription and cohesin localization. Chromatin immunoprecipitation followed by hybridization to a microarray (ChIP chip) indicated that transcript elongation into cohesin association sites results in the local disassociation of cohesin. Once transcription is halted, cohesin can reassociate with its original sites, independent of DNA replication and the cohesin loading factor Scc2, although cohesin association with chromosomes in G 2 /M is not functional for cohesion. A computer program was developed to systematically identify differences between two ChIP chip data sets. Our results are consistent with a model for cohesin association in which (i) a portion of cohesin can be dynamically loaded and unloaded to accommodate transcription and (ii) the cohesin complex has preferences for features of chromatin that are a reflection of the local transcriptional status. Taken together, our results suggest that cohesion may be degraded by transcription.Dividing cells must ensure that their chromosomes are copied exactly once and that new cells receive exactly a single copy of each chromosome. Failure to do so can result in aneuploidy, an abnormal number of chromosomes that typically leads to developmental abnormalities or cell death. During DNA replication, sister chromatids are cohered and cohesion is maintained until the metaphase-to-anaphase transition (28, 42). Sister chromatid cohesion facilitates the biorientation of chromosomes along the metaphase spindle and resists the tendency of microtubules to prematurely separate chromosomes once bipolar attachments are established (38). Cohesin also contributes to DNA repair (37, 44) and condensation (15).In eukaryotic cells, cohesion is mediated by several evolutionarily conserved proteins. The complex itself is composed of two SMC (structural maintenance of chromosomes) subunits, Smc1 and Smc3, and two non-SMC subunits, Mcd1/Scc1 and Scc3. Together, these subunits form a large ring-shaped complex that is essential for cohesion between sister chromatids (14, 16). The loading of cohesin onto chromosomes in G 1 is dependent on the Scc2/4 complex (5, 40). Cohesion is established during DNA replication (42). Cohesion is dissolved at the metaphase-to-anaphase transition by separase, which cleaves Mcd1, resulting in the movement of sister chromatids into separate daughter cells (41,43). Although the molecular structure of cohesin has been well studied, exactly how this ring-shaped complex interacts with DNA remains unc...
The first generation of biochemical studies of complex, iron-sulfur-cluster-containing [FeFe]-hydrogenases and Mo-nitrogenase were carried out on enzymes purified from Clostridium pasteurianum (strain W5). Previous studies suggested that two distinct [FeFe]-hydrogenases are expressed differentially under nitrogen-fixing and non-nitrogen-fixing conditions. As a result, the first characterized [FeFe]-hydrogenase (CpI) is presumed to have a primary role in central metabolism, recycling reduced electron carriers that accumulate during fermentation via proton reduction. A role for capturing reducing equivalents released as hydrogen during nitrogen fixation has been proposed for the second hydrogenase, CpII. Biochemical characterization of CpI and CpII indicated CpI has extremely high hydrogen production activity in comparison to CpII, while CpII has elevated hydrogen oxidation activity in comparison to CpI when assayed under the same conditions. This suggests that these enzymes have evolved a catalytic bias to support their respective physiological functions. Using the published genome of C. pasteurianum (strain W5) hydrogenase sequences were identified, including the already known [NiFe]-hydrogenase, CpI, and CpII sequences, and a third hydrogenase, CpIII was identified in the genome as well. Quantitative real-time PCR experiments were performed in order to analyze transcript abundance of the hydrogenases under diazotrophic and non-diazotrophic growth conditions. There is a markedly reduced level of CpI gene expression together with concomitant increases in CpII gene expression under nitrogen-fixing conditions. Structure-based analyses of the CpI and CpII sequences reveal variations in their catalytic sites that may contribute to their alternative physiological roles. This work demonstrates that the physiological roles of CpI and CpII are to evolve and to consume hydrogen, respectively, in concurrence with their catalytic activities in vitro, with CpII capturing excess reducing equivalents under nitrogen fixation conditions. Comparison of the primary sequences of CpI and CpII and their homologs provides an initial basis for identifying key structural determinants that modulate hydrogen production and hydrogen oxidation activities.
The central histone H3/H4 chaperone Asf1 comprises a highly conserved globular core and a divergent C-terminal tail. While the function and structure of the Asf1 core are well known, the function of the tail is less well understood. Here, we have explored the role of the yeast (yAsf1) and human (hAsf1a and hAsf1b) Asf1 tails in Saccharomyces cerevisiae. We show, using a photoreactive, unnatural amino acid, that Asf1 tail residue 210 cross-links to histone H3 in vivo and, further, that loss of C-terminal tail residues 211 to 279 weakens yAsf1-histone binding affinity in vitro nearly 200-fold. Via several yAsf1 C-terminal truncations and yeast-human chimeric proteins, we found that truncations at residue 210 increase transcriptional silencing and that the hAsf1a tail partially substitutes for full-length yAsf1 with respect to silencing but that full-length hAsf1b is a better overall substitute for full-length yAsf1. In addition, we show that the C-terminal tail of Asf1 is phosphorylated at T270 in yeast. Loss of this phosphorylation site does not prevent coimmunoprecipitation of yAsf1 and Rad53 from yeast extracts, whereas amino acid residue substitutions at the Asf1-histone H3/H4 interface do. Finally, we show that residue substitutions in yAsf1 near the CAF-1/HIRA interface also influence yAsf1's function in silencing.
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