Sulfide oxidation in the phototrophic purple sulfur bacterium Chromatium vinosum D (DSMZ 180(T)) was studied by insertional inactivation of the fccAB genes, which encode flavocytochrome c, a protein that exhibits sulfide dehydrogenase activity in vitro. Flavocytochrome c is located in the periplasmic space as shown by a PhoA fusion to the signal peptide of the hemoprotein subunit. The genotype of the flavocytochrome-c-deficient Chr. vinosum strain FD1 was verified by Southern hybridization and PCR, and the absence of flavocytochrome c in the mutant was proven at the protein level. The oxidation of thiosulfate and intracellular sulfur by the flavocytochrome-c-deficient mutant was comparable to that of the wild-type. Disruption of the fccAB genes did not have any significant effect on the sulfide-oxidizing ability of the cells, showing that flavocytochrome c is not essential for oxidation of sulfide to intracellular sulfur and indicating the presence of a distinct sulfide-oxidizing system. In accordance with these results, Chr. vinosum extracts catalyzed electron transfer from sulfide to externally added duroquinone, indicating the presence of the enzyme sulfide:quinone oxidoreductase (EC 1.8.5.-). Further investigations showed that the sulfide:quinone oxidoreductase activity was sensitive to heat and to quinone analogue inhibitors. The enzyme is strictly membrane-bound and is constitutively expressed. The presence of sulfide:quinone oxidoreductase points to a connection of sulfide oxidation to the membrane electron transport system at the level of the quinone pool in Chr. vinosum.

Experimental evidence suggests that most members of class A G-protein coupled receptors (GPCRs) can form homomers and heteromers in addition to functioning as single monomers. In particular, serotonin (5-HT) receptors were shown to homodimerize and heterodimerize with other GPCRs, although the details and the physiological role of the oligomerization has not yet been fully elucidated. Here we used computational modeling of the 5-HT 1A receptor monomer and dimer to predict residues important for dimerization. Based on these results, we carried out rationally designed site-directed mutagenesis. The ability of the mutants to dimerize was evaluated using different FRET-based approaches. The reduced levels of acceptor photobleaching-Fö rster resonance energy transfer (FRET) and the lower number of monomers participating in oligomers, as assessed by lux-FRET, confirmed the decreased ability of the mutants to dimerize and the involvement of the predicted contacts (Trp175 4.64 , Tyr198 5.41 , Arg151 4.40 , and Arg152 4.41 ) at the interface. This information was reintroduced as constraints for computational protein-protein docking to obtain a high-quality dimer model. Analysis of the refined model as well as molecular dynamics simulations of wild-type (WT) and mutant dimers revealed compensating interactions in dimers composed of WT and W175A mutant. This provides an explanation for the requirement of mutations of Trp175 4.64 in both homomers for disrupting dimerization. Our iterative computational-experimental study demonstrates that transmembrane domains TM4/ TM5 can form an interaction interface in 5-HT 1A receptor dimers and indicates that specific amino acid interactions maintain this interface. The mutants and the optimized model of the dimer structure may be used in functional studies of serotonin dimers.

Nitric oxide (NO) is produced by different isoforms of nitric oxide synthases (NOSs) and operates as a mediator of important cell signaling pathways, such as the cGMP signaling cascade. Another mechanism by which NO exerts biological effects is mediated through S-nitrosation of target proteins. To explore thiol-based protein modifications in a situation of defined nitrosative stress, we used a transgenic mouse model with cardiac specific overexpression of inducible nitric oxide synthase (iNOS) and concomitant myoglobin deficiency (iNOS). In comparison with the wild type hearts, protein glutathiolation detected by immunoblotting was significantly enhanced in iNOS ؉ /myo ؊/؊ hearts, whereas protein S-nitrosation as measured by the biotin switch assay and two-dimensional PAGE revealed that nearly all of the detected proteins (ϳ60) remained unchanged with the exception of three proteins. Tandem mass spectrometry revealed these proteins to be peroxiredoxins (Prxs), which are known to possess peroxidase activity, whereby hydrogen peroxide, peroxynitrite, and a wide range of organic hydroperoxides are reduced and detoxified. Immunoblotting with specific antibodies revealed up-regulation of Prx VI in the iNOS ؉ /myo ؊/؊ hearts, whereas expression of Prx II and Prx III remained unchanged. Furthermore, the analysis of the cardiac S-nitrososubproteome identified several new proteins possibly being involved in NO-signaling pathways. Our data indicate that S-nitrosation and glutathiolation of cardiac proteins may contribute to the phenotype of NO-induced heart failure. The upregulation of antioxidant proteins like Prx VI appears to be an additional mechanism to antagonize an excess of reactive oxygen/nitrogen species. Furthermore, S-nitrosation of Prxs may serve a new function in the signaling cascade of nitrosative stress. Nitric oxide (NO)2 is an important mediator in the regulation of cardiovascular physiology being involved in the modulation of vascular tone, cardiac contraction, relaxation, and energetics (1, 2). To fulfill these diverse functions in the heart, NO is synthesized by different nitric oxide synthase (NOS) isozymes, whereby the constitutively expressed endothelial NOS and neuronal NOS isozymes are restricted to defined subcellular microdomains requiring calcium and calmodulin for their activation. The expression of inducible NOS (iNOS) is stimulated in response to inflammation and cytokine production also in cardiomyocytes (3). In comparison with the other isozymes, the activation of iNOS is calcium/calmodulin-independent, and larger quantities of NO are produced. Although iNOS releases high levels of NO, which in cardiac myocytes is known to induce apoptosis (4), inhibit the respiratory chain (5), and induce a negative inotropic effect even under basal conditions (6), transgenic mice with cardiospecific overexpression of iNOS do not develop heart failure (7), calling into question the concept that cardiac iNOS induction may be an important factor in the pathogenesis of heart failure (8). However, double mu...