Ferrihydrite-dependent growth of Sulfurospirillum deleyianum through electron transfer via sulfur cycling

Observations in enrichment cultures of ferric iron-reducing bacteria indicated that ferrihydrite was reduced to ferrous iron minerals via sulfur cycling with sulfide as the reductant. Ferric iron reduction via sulfur cycling was investigated in more detail with Sulfurospirillum deleyianum, which can utilize sulfur or thiosulfate as an electron acceptor. In the presence of cysteine (0.5 or 2 mM) as the sole sulfur source, no (microbial) reduction of ferrihydrite or ferric citrate was observed, indicating that S. deleyianum is unable to use ferric iron as an immediate electron acceptor. However, with thiosulfate at a low concentration (0.05 mM), growth with ferrihydrite (6 mM) was possible and sulfur was cycled up to 60 times. Also, spatially distant ferrihydrite in agar cultures was reduced via diffusible sulfur species. Due to the low concentrations of thiosulfate, S. deleyianum produced only small amounts of sulfide. Obviously, sulfide delivered electrons to ferrihydrite with no or only little precipitation of black iron sulfides. Ferrous iron and oxidized sulfur species were produced instead, and the latter served again as the electron acceptor. These oxidized sulfur species have not yet been identified. However, sulfate and sulfite cannot be major products of ferrihydrite-dependent sulfide oxidation, since neither compound can serve as an electron acceptor for S. deleyianum. Instead, sulfur (elemental S or polysulfides) and/or thiosulfate as oxidized products could complete a sulfur cycle-mediated reduction of ferrihydrite.     

Sulfur Species as Redox Partners and Electron Shuttles for Ferrihydrite Reduction by Sulfurospirillum deleyianum

Iron(III) (oxyhydr)oxides can represent the dominant microbial electron acceptors under anoxic conditions in many aquatic environments, which makes understanding the mechanisms and processes regulating their dissolution and transformation particularly important. In a previous laboratory-based study, it has been shown that 0.05 mM thiosulfate can reduce 6 mM ferrihydrite indirectly via enzymatic reduction of thiosulfate to sulfide by the sulfur-reducing bacterium Sulfurospirillum deleyianum, followed by abiotic reduction of ferrihydrite coupled to reoxidation of sulfide. Thiosulfate, elemental sulfur, and polysulfides were proposed as reoxidized sulfur species functioning as electron shuttles. However, the exact electron transfer pathway remained unknown. Here, we present a detailed analysis of the sulfur species involved. Apart from thiosulfate, substoichiometric amounts of sulfite, tetrathionate, sulfide, or polysulfides also initiated ferrihydrite reduction. The portion of thiosulfate produced during abiotic ferrihydrite-dependent reoxidation of sulfide was about 10% of the total sulfur at maximum. The main abiotic oxidation product was elemental sulfur attached to the iron mineral surface, which indicates that direct contact between microorganisms and ferrihydrite is necessary to maintain the iron reduction process. Polysulfides were not detected in the liquid phase. Minor amounts were found associated either with microorganisms or the mineral phase. The abiotic oxidation of sulfide in the reaction with ferrihydrite was identified as rate determining. Cysteine, added as a sulfur source and a reducing agent, also led to abiotic ferrihydrite reduction and therefore should be eliminated when sulfur redox reactions are investigated. Overall, we could demonstrate the large impact of intermediate sulfur species on biogeochemical iron transformations.     

Complete genome sequence of Sulfurospirillum deleyianum type strain (5175)

Sulfurospirillum deleyianum Schumacher et al. 1993 is the type species of the genus Sulfurospirillum. S. deleyianum is a model organism for studying sulfur reduction and dissimilatory nitrate reduction as an energy source for growth. Also, it is a prominent model organism for studying the structural and functional characteristics of cytochrome c nitrite reductase. Here, we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of the genus Sulfurospirillum. The 2,306,351 bp long genome with its 2,291 protein-coding and 52 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Anodes Stimulate Anaerobic Toluene Degradation via Sulfur Cycling in Marine Sediments

Hydrocarbons released during oil spills are persistent in marine sediments due to the absence of suitable electron acceptors below the oxic zone. Here, we investigated an alternative bioremediation strategy to remove toluene, a model monoaromatic hydrocarbon, using a bioanode. Bioelectrochemical reactors were inoculated with sediment collected from a hydrocarbon-contaminated marine site, and anodes were polarized at 0 mV and +300 mV (versus an Ag/AgCl [3 M KCl] reference electrode). The degradation of toluene was directly linked to current generation of up to 301 mA m⁻² and 431 mA m⁻² for the bioanodes polarized at 0 mV and +300 mV, respectively. Peak currents decreased over time even after periodic spiking with toluene. The monitoring of sulfate concentrations during bioelectrochemical experiments suggested that sulfur metabolism was involved in toluene degradation at bioanodes. 16S rRNA gene-based Illumina sequencing of the bulk anolyte and anode samples revealed enrichment with electrocatalytically active microorganisms, toluene degraders, and sulfate-reducing microorganisms. Quantitative PCR targeting the α-subunit of the dissimilatory sulfite reductase (encoded by dsrA) and the α-subunit of the benzylsuccinate synthase (encoded by bssA) confirmed these findings. In particular, members of the family Desulfobulbaceae were enriched concomitantly with current production and toluene degradation. Based on these observations, we propose two mechanisms for bioelectrochemical toluene degradation: (i) direct electron transfer to the anode and/or (ii) sulfide-mediated electron transfer.     

Growth substrate dependent localization of tetrachloroethene reductive dehalogenase in Sulfurospirillum multivorans

Sulfurospirillum multivorans is a dehalorespiring organism, which is able to utilize tetrachloroethene as terminal electron acceptor in an anaerobic respiratory chain. The localization of the tetrachloroethene reductive dehalogenase in dependence on different growth substrates was studied using the freeze-fracture replica immunogold labeling technique. When the cells were grown with pyruvate plus fumarate, a major part of the enzyme was either localized in the cytoplasm or membrane associated facing the cytoplasm. In cells grown on pyruvate or formate as electron donors and tetrachloroethene as electron acceptor, most of the enzyme was detected at the periplasmic side of the cytoplasmic membrane. These results were confirmed by immunoblots of the enzyme with and without the twin arginine leader peptide. Trichloroethene exhibited the same effect on the enzyme localization as tetrachloroethene. The data indicated that the localization of the enzyme was dependent on the electron acceptor utilized.     

Endogenous Phenazine Antibiotics Promote Anaerobic Survival of Pseudomonas aeruginosa via Extracellular Electron Transfer

Antibiotics are increasingly recognized as having other, important physiological functions for the cells that produce them. An example of this is the effect that phenazines have on signaling and community development for Pseudomonas aeruginosa (L. E. Dietrich, T. K. Teal, A. Price-Whelan, and D. K. Newman, Science 321:1203-1206, 2008). Here we show that phenazine-facilitated electron transfer to poised-potential electrodes promotes anaerobic survival but not growth of Pseudomonas aeruginosa PA14 under conditions of oxidant limitation. Other electron shuttles that are reduced but not made by PA14 do not facilitate survival, suggesting that the survival effect is specific to endogenous phenazines.Phenazines have long been recognized for their redox properties. While most attention concerning their redox activity has focused on their role in generating reactive oxygen species in the context of infection (13, 14, 19, 22), as early as 1931, Friedheim hypothesized that phenazine reduction might benefit producer cells as an alternative respiratory pigment (12). Several years ago, our group suggested that the context in which this might be most important would be in biofilms, where cell densities are high and access to oxidants is limited (16, 31); consistent with this, we recently showed that mutants unable to produce phenazines are defective in community development (8). While this phenotype is likely due to many factors, including a signaling function for phenazines in later stages of growth (9), given the 1931 hypothesis by Friedheim (12) and our related recent work demonstrating that phenazines control redox homeostasis in Pseudomonas aeruginosa (30), we reasoned that phenazines might contribute to the survival of cells experiencing oxidant limitation.As a first step toward testing this, we investigated the effect of redox-active small molecules on anaerobic survival of P. aeruginosa PA14 in stationary-phase planktonic cultures. We justified beginning with planktonic cultures rather than biofilms based on previous studies which have suggested that cells in stationary-phase planktonic culture physiologically resemble cells in established biofilms (15, 35, 38). Moreover, by working with cells in planktonic cultures, we could build on voltammetric methods that had previously been used to determine how metabolism changes in Escherichia coli in the presence of the synthetic redox-shuttle ferricyanide (36). Similar voltammetric approaches have also been used to study how phenazines (32, 33) and structurally related flavins (23) mediate power generation by microbial fuel cells.We assembled bulk electrolysis-based glass bioreactors housed within an O₂- and H₂-free glove box (MBraun) and controlled by a multichannel potentiostat (series G 300; Gamry) outside the glove box. Each bioreactor held a graphite rod working electrode (Alfa Aesar) with an operating surface area of 6 cm², a Ag/AgCl reference electrode (RE-5B; BASi) with a constant potential of +207 mV versus that of the normal hydrogen electrode (NHE), and about 100 ml MOPS (morpholinepropanesulfonic acid) culture medium (100 mM MOPS at pH 7.2, 93 mM NH₄Cl, 43 mM NaCl, 2.2 mM KH₂PO₄, 1 mM MgSO₄, 5.0 μM FeCl₃) (modified from reference 29). The bioreactor was joined by a fritted glass junction to a small side chamber, in which a Pt counter electrode made from Pt mesh (Alfa Aesar) soldered to a copper wire completed the circuit. In order to selectively examine different redox-active small molecules, we used the phenazine-null mutant of PA14 (Δphz) which is deleted in its two phenazine biosynthetic operons (9).We began by focusing on three endogenous phenazines—pyocyanin (PYO), phenazine-1-carboxylic acid (PCA), and 1-hydroxyphenazine (1-OHPHZ)—that are known to be excreted by PA14 during stationary-phase growth cycle in laboratory cultures (9, 17). We harvested cells from cultures grown aerobically on Luria-Bertani (LB; Fisher Scientific) medium at 37°C and concentrated and resuspended them in anaerobic MOPS medium at 10⁹ CFU/ml. These resuspended cells were incubated in bioreactors over a period of 7 days at 30°C. To perform the survival experiments, we incubated dense suspensions (10⁹ cells/ml) in the MOPS medium containing 20 mM d-glucose to ensure excess electron donor, added ∼90 μM phenazine (PYO, PCA, or 1-OHPHZ), and poised the working electrode at +200 mV versus that of the NHE to make certain it was just high enough to efficiently oxidize bacterially reduced phenazine but not other medium components (e.g., d-glucose). To confirm the poised potential was appropriate to ensure that phenazine was the sole reversible redox-active component, we compared the cyclic voltammetries (CV) of Δphz cultures with or without phenazine at the time point immediately prior to the start of the survival test. Using the CV method described in detail previously (39), we found that Δphz cultures supplemented with phenazine exhibited single anodic (oxidation) and cathodic (reduction) peaks; these peaks were absent when phenazine was not present. This is illustrated in Fig. ​Fig.11 for PYO. For Δphz cultures supplemented with phenazine, we collected supernatants at the beginning and the end of each survival test for high-performance liquid chromatography (HPLC) analyses using a previously developed method (9). In both cases, HPLC samples yielded the same single phenazine peak with characteristic peak size, implying that degradation did not occur in these experiments. Throughout the incubation period, we continuously recorded the anodic (oxidation) current as well as the charge transferred due to the oxidation of an electrochemically active component(s) at the working electrode. Periodically, we sampled to measure viability by means of counting the number of CFU on LB agar (11).Open in a separate windowFIG. 1.Representative cyclic voltammetry (CV) of Δphz mutant cultures of P. aeruginosa PA14 incubated anaerobically in 100 ml MOPS medium containing 20 mM glucose, supplemented with 90 μM PYO (dark trace) or no PYO (light trace). PYO is the only electrochemically active component with single anodic (oxidation) and cathodic (reduction) peaks characteristic of itself. CV experiments were performed at 100 mV/s, with electrodes consisting of a stationary gold disk working electrode (BASi), an Ag/AgCl reference electrode, and a Pt counter electrode.We observed that the Δphz mutant maintained a constant viable cell number at the original 10⁹ CFU/ml over 7 days, characteristic of survival but not growth (Fig. ​(Fig.2).2). In contrast, when we incubated the Δphz mutant in the bioreactors without adding phenazine or applying a potential or both, the cells sustained their viability up to day 3 and then dropped logarithmically down to 0.1 to 1% of the original 10⁹ CFU/ml by day 7 (Fig. ​(Fig.2).2). Our electrochemical observations were in agreement with the number of CFU results. Without phenazine supplementation, we observed a constant anodic current in the range of 5 to 10 μA with the poised potential, most likely reflecting a background current due to slow oxidation of medium components, which was not able to help Δphz survive over 7 days. In the presence of phenazine, however, the anodic current increased from the background level to 80 ± 10 μA for PYO and PCA and 45 ± 10 μA for 1-OHPHZ within 2 h and stayed at the high current levels with slow decay (less than 20%) throughout the incubation period. The slow current decay was likely due to electrode fouling (23) and/or the accumulation of toxic metabolic by-products in the batch reactors over time. The facile reversibility of redox-active phenazines, which are reduced within the bacterial cell and oxidized outside the cell by the working electrode, led to the high current readings and was key to Δphz survival.Open in a separate windowFIG. 2.PYO (a), PCA (b), and 1-OHPHZ (c) function as electron shuttles (▪) to promote anaerobic survival of the Δphz mutant of P. aeruginosa PA14 when cells are incubated anaerobically in MOPS-buffered medium containing 20 mM d-glucose and ∼90 μM phenazine (PYO, PCA, or 1-OHPHZ) and with the graphite rod working electrode poised at +0.2 V versus that of the NHE. Survival was determined by measuring the number of CFU on LB agar plates. The number of CFU of Δphz anaerobic incubations without phenazine (▿), poised potential (▵), or both (⋄) served as a control. Error bars represent standard deviations from at least triplicate samples in each experimental set. Plots represent results from at least three independent experiments.We then estimated the average number of redox cycles (defined as “a”) for each phenazine molecule, which is known to undergo two-electron oxidation-reduction (39), throughout the 7-day incubation based on the equation adapted from Faraday''s law for bulk electrolysis (1), Q = 2FN = 2F(acv), with c as the phenazine concentration (in M), v as reaction volume (in liters), F as Faraday''s constant (96,485 C/mole), N (which equals acv) as the amount of phenazine (in moles) involved in the electrolysis, and Q (in coulombs) as the net charge quantity associated with the electrochemical oxidation of reduced phenazine during electrolysis (by subtracting the background charge without phenazine from the total charge passed with phenazine). By recording Q, and knowing c and v, we calculated the number of redox cycles over 7 days for PYO, PCA, and 1-OHPHZ to be 31, 22, and 14, respectively. Moreover, each of the three phenazines showed the color characteristic of its oxidized form during redox cycling rather than that of its reduced form, which was apparent when cycling was prevented by not applying the poised potential (39); this indicated that intracellular phenazine reduction was the rate-limiting step of each redox cycle. In addition, we observed the following correlation between the reaction kinetics and phenazine thermodynamic properties: both the phenazine reduction potential (Table ​(Table1)1) and the intracellular reduction rate decreased in the order PYO > PCA > 1-OHPHZ. In summary, despite different electron-shuttling efficiencies, all three phenazines supported Δphz survival equally well within the testing period by acting as electron acceptors (Fig. ​(Fig.22).

TABLE 1.

Properties and results summarized from experiments for testing the roles of endogenous phenazines and other type redox-active compounds in promoting anaerobic survival of P. aeruginosa

Coral-Associated Bacteria and Their Role in the Biogeochemical Cycling of Sulfur

Marine bacteria play a central role in the degradation of dimethylsulfoniopropionate (DMSP) to dimethyl sulfide (DMS) and acrylic acid, DMS being critical to cloud formation and thereby cooling effects on the climate. High concentrations of DMSP and DMS have been reported in scleractinian coral tissues although, to date, there have been no investigations into the influence of these organic sulfur compounds on coral-associated bacteria. Two coral species, Montipora aequituberculata and Acropora millepora, were sampled and their bacterial communities were characterized by both culture-dependent and molecular techniques. Four genera, Roseobacter, Spongiobacter, Vibrio, and Alteromonas, which were isolated on media with either DMSP or DMS as the sole carbon source, comprised the majority of clones retrieved from coral mucus and tissue 16S rRNA gene clone libraries. Clones affiliated with Roseobacter sp. constituted 28% of the M. aequituberculata tissue libraries, while 59% of the clones from the A. millepora libraries were affiliated with sequences related to the Spongiobacter genus. Vibrio spp. were commonly isolated from DMS and acrylic acid enrichments and were also present in 16S rRNA gene libraries from coral mucus, suggesting that under “normal” environmental conditions, they are a natural component of coral-associated communities. Genes homologous to dddD, and dddL, previously implicated in DMSP degradation, were also characterized from isolated strains, confirming that bacteria associated with corals have the potential to metabolize this sulfur compound when present in coral tissues. Our results demonstrate that DMSP, DMS, and acrylic acid potentially act as nutrient sources for coral-associated bacteria and that these sulfur compounds are likely to play a role in structuring bacterial communities in corals, with important consequences for the health of both corals and coral reef ecosystems.Dimethylsulfoniopropionate (DMSP) is an organic sulfur compound implicated in the formation of clouds via its cleavage product dimethyl sulfide (DMS) and therefore has the potential to exert major cooling effects on climate (9, 38). The production of DMSP is mainly restricted to a few classes of marine macro- and microalgae (27, 68), with the main producers being phytoplankton species belonging to prymnesiophyte and dinoflagellate taxa (28, 62, 67). Recently, significant concentrations of DMSP and DMS have been recorded in association with animals that harbor symbiotic algae such as scleractinian corals and giant clams (7, 8, 68), raising questions about the role of coral reefs in sulfur cycling. The densities of symbiotic dinoflagellates (genus Symbiodinium, commonly known as zooxanthellae) in coral tissues are similar to those recorded for dinoflagellates in phytoplankton blooms (11, 68). Since dinoflagellates are among the most significant producers of DMSP and high intracellular concentrations of DMSP have been found in both cultured zooxanthellae (26) and scleractinian corals (6-8, 25), these observations suggest that endosymbiotic zooxanthellae have an integral role in sulfur cycling in oligotrophic reef waters.Most of the DMSP produced by planktonic dinoflagellates is exuded into the surrounding water, where it is degraded by bacteria via two possible pathways: the first one converts a large fraction (ca. 75%) of dissolved DMSP to methylmercaptopropionate, which is subsequently incorporated into the biomass of microbial cells (22, 27, 66). The second pathway transforms the remaining part of the dissolved DMSP to equimolar concentrations of DMS and acrylic acid (43, 66, 72). This metabolic pathway for DMSP degradation has been identified in the alphaproteobacterial species Sulfitobacter sp. and the enzyme involved (DMSP-dependent DMS lyase [DddL]) characterized (10). Another pathway for DMS formation (without production of acrylate) has been described for Marinomonas sp. and the gene responsible, dddD, identified. In addition, the protein DddR has been directly implicated in the regulation of the gene encoding DddD (66). The DMS produced by these enzymes are then released into the surrounding water (27). Prior to the 1980s, diffusion of supersaturated DMS from the oceans to the atmosphere was thought to be the major removal pathway of this compound from the oceans (35, 72). More recently, however, it has been estimated that between 50 and 80% of the DMS produced by DMSP-degrading bacteria is degraded directly by other types of bacteria (58, 59), although the populations and metabolic pathways involved in the degradation of DMS are still poorly understood.Coral-associated bacterial communities are known to be diverse and highly abundant (12, 30, 48, 49, 52). These dynamic communities exploit a number of habitats associated with corals, including mucus on coral surfaces (48), intracellular niches within coral tissues (3, 16, 45, 47, 52), spaces within coral skeletons (15, 51), and seawater surrounding corals (16, 61). Each of these habitats is believed to harbor different bacterial populations (4, 52). Despite high bacterial diversity, corals have been reported to harbor species-specific microbial communities for beneficial effects; however, their role in coral health is poorly understood (47-50). In coral reef environments, bacteria are dependent upon organic compounds produced by photoautotrophic organisms such as endosymbiotic zooxanthellae (48); therefore, photosynthates translocated to coral tissues and mucus may determine microbial communities closely associated with corals (48, 52). The high levels of DMSP and DMS produced by corals, coupled with the dependence of DMSP and DMS conversion on processes typically involving bacteria, suggest that corals are likely to harbor bacterial species involved in the cycling of these compounds. To investigate the potential of the organosulfur compound DMSP and its breakdown products, DMS and acrylic acid, to drive coral-associated microbial communities, we used these compounds as sole carbon sources to isolate bacteria from two coral species (Montipora aequituberculata and Acropora millepora) and then directly compared these microbial communities with coral-associated microbiota identified using culture-independent analyses. Genes implicated in the metabolism of DMSP were also characterized from isolated strains, confirming that bacteria associated with corals have the potential to metabolize organic sulfur compounds present in coral tissues.     

Erv1 and Cytochrome c Mediate Rapid Electron Transfer via A Collision-Type Interaction

Being essential for oxidative protein folding in the mitochondrial intermembrane space, the mitochondrial disulfide relay relies on the electron transfer (ET) from the sulfhydryl oxidase Erv1 to cytochrome c (Cc). Using solution NMR spectroscopy, we demonstrate that while the yeast Cc-Erv1 system is functionally active, no observable binding of the protein partners takes place. The transient interaction between Erv1 and Cc can be rationalized by molecular modeling, suggesting that a large surface area of Erv1 can sustain a fast ET to Cc via a collision-type mechanism, without the need for a canonical protein complex formation. We suggest that, by preventing the direct ET to molecular oxygen (O₂), the collision-type Cc-Erv1 interaction plays a role in protecting the organism against reactive oxygen species.     

醌血红素蛋白的电子传递

醌蛋白是以吡咯喹啉醌（PQQ）及其结构类似物为辅酶的一类氧化还原酶。醌血红素蛋白是以PQQ和一个或多个血红素作为辅助因子的醌蛋白,包括醌血红素蛋白醇脱氢酶和醌血红素蛋白胺脱氢酶。简要综述了醌血红素蛋白的结构特点,在分子内从PQQ到血红素的电子传递,以及醌血红素蛋白与蛋白之间的分子间的电子传递。     

Escherichia coli SufE Sulfur Transfer Protein Modulates the SufS Cysteine Desulfurase through Allosteric Conformational Dynamics

Fe-S clusters are critical metallocofactors required for cell function. Fe-S cluster biogenesis is carried out by assembly machinery consisting of multiple proteins. Fe-S cluster biogenesis proteins work together to mobilize sulfide and iron, form the nascent cluster, traffic the cluster to target metalloproteins, and regulate the assembly machinery in response to cellular Fe-S cluster demand. A complex series of protein-protein interactions is required for the assembly machinery to function properly. Despite considerable progress in obtaining static three-dimensional structures of the assembly proteins, little is known about transient protein-protein interactions during cluster assembly or the role of protein dynamics in the cluster assembly process. The Escherichia coli cysteine desulfurase SufS (EC 2.8.1.7) and its accessory protein SufE work together to mobilize persulfide from l-cysteine, which is then donated to the SufB Fe-S cluster scaffold. Here we use amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize SufS-SufE interactions and protein dynamics in solution. HDX-MS analysis shows that SufE binds near the SufS active site to accept persulfide from Cys-364. Furthermore, SufE binding initiates allosteric changes in other parts of the SufS structure that likely affect SufS catalysis and alter SufS monomer-monomer interactions. SufE enhances the initial l-cysteine substrate binding to SufS and formation of the external aldimine with pyridoxal phosphate required for early steps in SufS catalysis. Together, these results provide a new picture of the SufS-SufE sulfur transferase pathway and suggest a more active role for SufE in promoting the SufS cysteine desulfurase reaction for Fe-S cluster assembly.     

Cellular Metabolites Enhance the Light Sensitivity of Arabidopsis Cryptochrome through Alternate Electron Transfer Pathways

Cryptochromes are blue light receptors with multiple signaling roles in plants and animals. Plant cryptochrome (cry1 and cry2) biological activity has been linked to flavin photoreduction via an electron transport chain comprising three evolutionarily conserved tryptophan residues known as the Trp triad. Recently, it has been reported that cry2 Trp triad mutants, which fail to undergo photoreduction in vitro, nonetheless show biological activity in vivo, raising the possibility of alternate signaling pathways. Here, we show that Arabidopsis thaliana cry2 proteins containing Trp triad mutations indeed undergo robust photoreduction in living cultured insect cells. UV/Vis and electron paramagnetic resonance spectroscopy resolves the discrepancy between in vivo and in vitro photochemical activity, as small metabolites, including NADPH, NADH, and ATP, were found to promote cry photoreduction even in mutants lacking the classic Trp triad electron transfer chain. These metabolites facilitate alternate electron transfer pathways and increase light-induced radical pair formation. We conclude that cryptochrome activation is consistent with a mechanism of light-induced electron transfer followed by flavin photoreduction in vivo. We further conclude that in vivo modulation by cellular compounds represents a feature of the cryptochrome signaling mechanism that has important consequences for light responsivity and activation.     

Suppression of Electron Transfer to Dioxygen by Charge Transfer and Electron Transfer Complexes in the FAD-dependent Reductase Component of Toluene Dioxygenase

The three-component toluene dioxygenase system consists of an FAD-containing reductase, a Rieske-type [2Fe-2S] ferredoxin, and a Rieske-type dioxygenase. The task of the FAD-containing reductase is to shuttle electrons from NADH to the ferredoxin, a reaction the enzyme has to catalyze in the presence of dioxygen. We investigated the kinetics of the reductase in the reductive and oxidative half-reaction and detected a stable charge transfer complex between the reduced reductase and NAD⁺ at the end of the reductive half-reaction, which is substantially less reactive toward dioxygen than the reduced reductase in the absence of NAD⁺. A plausible reason for the low reactivity toward dioxygen is revealed by the crystal structure of the complex between NAD⁺ and reduced reductase, which shows that the nicotinamide ring and the protein matrix shield the reactive C4a position of the isoalloxazine ring and force the tricycle into an atypical planar conformation, both factors disfavoring the reaction of the reduced flavin with dioxygen. A rapid electron transfer from the charge transfer complex to electron acceptors further reduces the risk of unwanted side reactions, and the crystal structure of a complex between the reductase and its cognate ferredoxin shows a short distance between the electron-donating and -accepting cofactors. Attraction between the two proteins is likely mediated by opposite charges at one large patch of the complex interface. The stability, specificity, and reactivity of the observed charge transfer and electron transfer complexes are thought to prevent the reaction of reductase_TOL with dioxygen and thus present a solution toward conflicting requirements.     

Unpaired Electron Migration between Aromatic and Sulfur Peptide Units

Cysteine thiyl radicals (Cys/S') were found capable of one-electron oxidation of tyrosine. Equilibration occurred, using Cys and Gly-Tyr, with an equilibrium constant of K₅ = 20 ± 4 at pH 9.15: Cys/S- + Tyr = Cys + Tyr/O

Hence the reduction potentials of these couples differ at pH 9.15 by E(Cys/S', Cys) - E(Tyr/O^r, Tyr) = 80 mV. Oxidation of Trp-Gly by Cys/S' was not detectable from pH 7 to 12. The methionyl radical cation (Met/S'N), formed via 'OH-attack on Met-Gly, reacts with Trp-Gly to generate the indolyl radical (Trp/N'). New results on intramolecular Trp/N' → Tyr/O' transitions indicate that the reaction requires direct contact between the two redox centers. Various possible pathways for migration of unpaired electrons between peptide units are compiled in a scheme.     

Growth of Thiobacillus ferrooxidans on Elemental Sulfur

Growth kinetics of Thiobacillus ferrooxidans in batch cultures, containing prills of elementary sulfur as the sole energy source, were studied by measuring the incorporation of radioactive phosphorus in free and adsorbed bacteria. The data obtained indicate an initial exponential growth of the attached bacteria until saturation of the susceptible surface was reached, followed by a linear release of free bacteria due to successive replication of a constant number of adsorbed bacteria. These adsorbed bacteria could continue replication provided the colonized prills were transferred to fresh medium each time the stationary phase was reached. The bacteria released from the prills were unable to multiply, and in the medium employed they lost viability with a half-life of 3.5 days. The spreading of the progeny on the surface was followed by staining the bacteria on the prills with crystal violet; this spreading was not uniform but seemed to proceed through distortions present in the surface. The specific growth rate of T. ferrooxidans ATCC 19859 was about 0.5 day⁻¹, both before and after saturation of the sulfur surface. The growth of adsorbed and free bacteria in medium containing both ferrous iron and elementary sulfur indicated that T. ferrooxidans can simultaneously utilize both energy sources.