Catalysis for chemical synthesis by cell-free monooxygenases necessitates an efficient and robust in situ regeneration system to supply the enzyme with reducing equivalents. We report on a novel approach to directly regenerate flavin-dependent monooxygenases. The organometallic complex [CpRh(bpy)(H(2)O)](2+) catalyzes the transhydrogenation reaction between formate and isoalloxazine-based cofactors such as FAD and FMN. Coupling this FADH(2) regeneration reaction to the FADH(2)-dependent styrene monooxygenase (StyA) resulted in a chemoenzymatic epoxidation reaction where the organometallic compound substitutes for the native reductase (StyB), the nicotinamide coenzyme (NAD), and an artificial NADH regeneration system such as formate dehydrogenase. Various styrene derivatives were converted into the essentially optically pure (S)-epoxides (ee > 98%). In addition, StyA was shown to be capable of performing sulfoxidation reactions. The productivity of the chemoenzymatic epoxidation reaction using 6.5 microM StyA reached up to 6.4 mM/h, corresponding to approximately 70% of a comparable fully enzymatic reaction using StyB, NADH, and formate dehydrogenase for regeneration. The coupling efficiency of the nonenzymatic regeneration reaction to enzymatic epoxidation was examined in detail, leading to an optimized reaction setup with minimized quenching of the electron supply for the epoxidation reaction. Thus, up to 60% of the reducing equivalents provided via [CpRh(bpy)(H(2)O)](2+) could be channeled into epoxide rather than hydrogen peroxide formation, allowing selective synthesis with high yields.

The pathogenicity of Vibrio cholerae is influenced by sodium ions which are actively extruded from the cell by the Na ؉ -translocating NADH:quinone oxidoreductase (Na ؉ -NQR). To study the function of the Na ؉ -NQR in the respiratory chain of V. cholerae, we examined the formation of organic radicals and superoxide in a wild-type strain and a mutant strain lacking the Na ؉ -NQR. Upon reduction with NADH, an organic radical was detected in native membranes by electron paramagnetic resonance spectroscopy which was assigned to ubisemiquinones generated by the Na ؉ -NQR. The radical concentration increased from 0.2 mM at 0.08 mM Na ؉ to 0.4 mM at 14.7 mM Na ؉ , indicating that the concentration of the coupling cation influences the redox state of the quinone pool in V. cholerae membranes. During respiration, V. cholerae cells produced extracellular superoxide with a specific activity of 10.2 nmol min ؊1 mg ؊1 in the wild type compared to 3.1 nmol min ؊1 mg ؊1in the NQR deletion strain. Raising the Na ؉ concentration from 0.1 to 5 mM increased the rate of superoxide formation in the wild-type V. cholerae strain by at least 70%. Rates of respiratory H 2 O 2 formation by wild-type V. cholerae cells (30.9 nmol min ؊1 mg ؊1 ) were threefold higher than rates observed with the mutant strain lacking the Na ؉ -NQR (9.7 nmol min ؊1 mg ؊1 ). Our study shows that environmental Na ؉ could stimulate ubisemiquinone formation by the Na ؉ -NQR and hereby enhance the production of reactive oxygen species formed during the autoxidation of reduced quinones.The gram-negative bacterium Vibrio cholerae naturally inhabits aquatic ecosystems, but some strains are able to colonize the human intestine, where they can cause the severe diarrheal disease cholera (10). As an adaptation for growth at high NaCl concentrations, V. cholerae expels sodium ions from the cytoplasm during respiration and establishes a sodium motive force across its inner membrane (12). This respiratory Na ϩ transport is catalyzed by the Na ϩ -translocating NADH:quinone oxidoreductase (Na ϩ -NQR), which consists of six subunits, NqrA to -F, and contains one Fe-S center, two covalently bound flavin mononucleotides, one non-covalently bound flavin adenine dinucleotide (FAD), one riboflavin, and ubiquinone-8 as prosthetic groups (5,17,41). Genome comparisons reveal that a Na ϩ -NQR is present in many pathogenic bacteria, indicating that pathogens may benefit from a sodium cycle for nutrient uptake or motility (13). The sodium motive force which is maintained by the Na ϩ -NQR strongly influences the production of virulence factors in Vibrio cholerae (14), and environmental Na ϩ is likely to be an important parameter during infection both as stimulus and as respiratory coupling ion (12). Loss of the Na ϩ -NQR, either by mutation or by chemical inhibition, results in altered virulence gene regulation in V. cholerae (14), but the putative link between sodium membrane energetics and virulence has not been identified yet. Superoxide (O 2Ϫ ) is an anionic free radical produced by ...

The enantiomeric siderophores pyochelin and enantiopyochelin of Pseudomonas aeruginosa and Pseudomonas protegens promote growth under iron limitation and activate transcription of their biosynthesis and uptake genes via the AraC-type regulator PchR. Here we investigated siderophore binding to PchR in vitro using fluorescence spectroscopy. A fusion of the N-terminal domain of P. aeruginosa PchR with maltose binding protein (MBP-PchR'PAO) bound iron-loaded (ferri-) pyochelin with an affinity (Kd) of 41 ± 5 μM. By contrast, no binding occurred with ferri-enantiopyochelin. Stereospecificity of a similar fusion protein of the P. protegens PchR (MBP-PchR'CHA0) was less pronounced. The Kd's of MBP-PchR'CHA0 for ferri-enantiopyochelin and ferri-pyochelin were 24 ± 5 and 40 ± 7 μM, respectively. None of the proteins interacted with the iron-free siderophore enantiomers, suggesting that transcriptional activation by PchR occurs only when the respective siderophore actively procures iron to the cell.

It is generally assumed that respiratory complexes exclusively use protons to energize the inner mitochondrial membrane. Here we show that oxidation of NADH by submitochondrial particles (SMPs) from the yeast Yarrowia lipolytica is coupled to protonophoreresistant Na + uptake, indicating that a redox-driven, primary Na + pump is operative in the inner mitochondrial membrane. By purification and reconstitution into proteoliposomes, a respiratory NADH dehydrogenase was identified which coupled NADH-dependent reduction of ubiquinone (1.4 lmol min -1 mg -1 ) to Na + translocation (2.0 lmol min -1 mg -1 ). NADH-driven Na + transport was sensitive towards rotenone, a specific inhibitor of complex I. We conclude that mitochondria from Y. lipolytica contain a NADH-driven Na + pump and propose that it represents the complex I of the respiratory chain. Our study indicates that energy conversion by mitochondria does not exclusively rely on the proton motive force but may benefit from the electrochemical Na + gradient established by complex I.

The Na؉ -translocating NADH:quinone oxidoreductase from Vibrio cholerae contains a single Fe-S cluster localized in subunit NqrF. Here we study the electronic properties of the Fe-S center in a truncated version of the NqrF subunit comprising only its ferredoxin-like Fe-S domain. Mö ssbauer spectroscopy of the Fe-S domain in the oxidized state is consistent with a binuclear Fe-S cluster with tetrahedral sulfur coordination by the cysteine residues Cys 70 , Cys 76 , Cys 79 , and Cys 111 . Important sequence motifs surrounding these cysteines are conserved in the Fe-S domain and in vertebrate-type ferredoxins. The magnetic circular dichroism spectra of the photochemically reduced Fe-S domain exhibit a striking similarity to the magnetic circular dichroism spectra of vertebrate-type ferredoxins required for the in vivo assembly of iron-sulfur clusters. This study reveals a novel function for vertebrate-type [2Fe-2S] clusters as redox cofactors in respiratory dehydrogenases.Iron-sulfur proteins are present in all domains of living organisms where they exhibit diverse functions like electron transport, catalysis, and sensing in regulatory processes (1). In some NADH-oxidizing, respiratory complexes, Fe-S centers accept electrons from flavin cofactors in an overall exergonic reaction that results in the reduction of quinone. This electron transfer reaction drives the uphill transport of protons or Na ϩ across the inner membrane of mitochondria or bacteria. The NADH:quinone oxidoreductase (Na ϩ -NQR) 1 from the human pathogen Vibrio cholerae maintains an electrochemical Na ϩ gradient across the inner bacterial membrane, which strongly influences the production of virulence factors (2). The Na ϩ -NQR consists of six subunits, NqrA-F, and contains one Fe-S center, two covalently bound FMNs, one non-covalently bound FAD, one riboflavin, and ubiquinone-8 as prosthetic groups (3-7). The NqrF subunit of the Na ϩ -NQR complex is anchored to the inner membrane and displays a clearly defined domain structure. The N-terminal Fe-S domain harbors the [2Fe-2S] cluster, while the binding sites for the non-covalently bound FAD and NADH are located in the C-terminal domain of NqrF. The initial oxidation of NADH by the NqrF subunit results in the two-electron reduction of the FAD followed by one-electron transfer steps to the [2Fe-2S] cluster in the Fe-S domain (7). Here we study the electronic properties of the [2Fe-2S] cluster in the isolated Fe-S domain of NqrF. A comparison of its amino acid sequence with sequences of [2Fe-2S] ferredoxins from vertebrates and plants reveals that the Fe-S domain is related to ferredoxins of the vertebrate-type family. Vertebrate-type ferredoxins are soluble redox carriers that accept electrons from specific NADH:ferredoxin reductases and deliver them to enzymatic systems catalyzing the hydroxylation of various compounds like steroids or camphor (8). The Fe-S domain exhibits highest sequence similarity to vertebrate-type ferredoxins required for the in vivo assembly of iron-sulfur clusters (ISC-type ...