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Background: Glioblastoma is the most common primary malignant tumor of the central nervous system that occurs in the spinal cord or brain. Pseudosynanceia melanostigma is a venomous stonefish in the Persian Gulf, which our knowledge about is little. This study’s goal is to investigate the toxicity of stonefish crude venom on mitochondria isolated from U87 cells. Methods: In the first stage, we extracted venom stonefish and then isolated mitochondria have exposed to different concentrations of venom. Finally, mitochondrial toxicity parameters (Succinate dehydrogenase (SDH) activity, Reactive oxygen species (ROS), cytochrome c release, Mitochondrial Membrane Potential (MMP), and mitochondrial swelling) have evaluated. Results: To determine mitochondrial parameters, we used 115, 230, and 460 µg/ml concentrations. The results of our study show that the venom of stonefish selectively increases upstream parameters of apoptosis such as mitochondrial swelling, cytochrome c release, MMP collapse and ROS. Conclusion: This study suggests that Pseudosynanceia melanostigma crude venom has selectively caused toxicity by increasing active mitochondrial oxygen radicals. This venom could potentially be a candidate for the treatment of glioblastoma.

Steroidal C7β alcohols and their respective esters have shown significant promise as neuroprotective and anti‐inflammatory agents to treat chronic neuronal damage like stroke, brain trauma, and cerebral ischemia. Since C7 is spatially far away from any functional groups that could direct C−H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450‐BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and β stereoselectivities, as well as high activities. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil‐Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high‐resolution X‐ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio‐ and stereoselectivity., One mutant, six targets: The regio‐ and stereoselective C−H activation for hydroxylation of six different steroids with formation of C7β alcohols was accomplished using a single P450 mutant evolved by protein library construction based on a special DNA assembly and cloning procedure. The C7β steroidal alcohols, not readily accessible by synthetic reagents or catalysts, are of intense interest as therapeutic drugs.

During photosynthesis, electrons are transferred between the cytochrome b6f complex and photosystem I. This is carried out by the protein plastocyanin in plant chloroplasts, or by either plastocyanin or cytochrome c6 in many cyanobacteria and eukaryotic algal species. There are three further cytochrome c6 homologs: cytochrome c6A in plants and green algae, and cytochromes c6B and c6C in cyanobacteria. The function of these proteins is unknown. Here, we present a comprehensive analysis of the evolutionary relationship between the members of the cytochrome c6 family in photosynthetic organisms. Our phylogenetic analyses show that cytochromes c6B and c6C are likely to be orthologs that arose from a duplication of cytochrome c6, but that there is no evidence for separate origins for cytochromes c6B and c6C. We therefore propose renaming cytochrome c6C as cytochrome c6B. We show that cytochrome c6A is likely to have arisen from cytochrome c6B rather than by an independent duplication of cytochrome c6, and present evidence for an independent origin of a protein with some of the features of cytochrome c6A in peridinin dinoflagellates. We conclude with a new comprehensive model of the evolution of the cytochrome c6 family which is an integral part of understanding the function of the enigmatic cytochrome c6 homologs.

Mitochondrial alternative oxidase (AOX) is predicted to be present in mitochondria of several invertebrate taxa including tardigrades. Independently of the reason concerning the enzyme occurrence in animal mitochondria, expression of AOX in human mitochondria is regarded as a potential therapeutic strategy. Till now, relevant data were obtained due to heterologous AOX expression in cells and animals without natively expressed AOX. Application of animals natively expressing AOX could importantly contribute to the research. Thus, we decided to investigate AOX activity in intact specimens of the tardigrade Hypsibius exemplaris. We observed that H. exemplaris specimens’ tolerance to the blockage of the mitochondrial respiratory chain (MRC) cytochrome pathway was diminished in the presence of AOX inhibitor and the inhibitor-sensitive respiration enabled the tardigrade respiration under condition of the blockage. Importantly, these observations correlated with relevant changes of the mitochondrial inner membrane potential (Δψ) detected in intact animals. Moreover, detection of AOX at protein level required the MRC cytochrome pathway blockage. Overall, we demonstrated that AOX activity in tardigrades can be monitored by the animals’ behavior observation as well as by measurement of intact specimens’ whole-body respiration and Δψ. Furthermore, it is also possible to check the impact of the MRC cytochrome pathway blockage on AOX level as well as AOX inhibition in the absence of the blockage on animal functioning. Thus, H. exemplaris could be consider as a whole-animal model suitable to study AOX.

Shewanella oneidensis MR‐1 strain with a higher cytoplasmic c‐di‐GMP level had a higher Fe(III) reduction rate and greater expression of genes that code for the proteins involved in the Mtr pathway. Single‐cell Raman microspectroscopy revealed a great increase of c‐type cytochromes in the high c‐di‐GMP strain as compared to MR‐1 wild‐type strain. c‐di‐GMP regulation system indirectly or directly positively regulates the expression of cytochromes involved in the extracellular electron transport (EET) in S. oneidensis., Summary Electrochemically active biofilms are capable of exchanging electrons with solid electron acceptors and have many energy and environmental applications such as bioelectricity generation and environmental remediation. The performance of electrochemically active biofilms is usually dependent on c‐type cytochromes, while biofilm development is controlled by a signal cascade mediated by the intracellular secondary messenger bis‐(3ʹ‐5ʹ) cyclic dimeric guanosine monophosphate (c‐di‐GMP). However, it is unclear whether there are any links between the c‐di‐GMP regulatory system and the expression of c‐type cytochromes. In this study, we constructed a S. oneidensis MR‐1 strain with a higher cytoplasmic c‐di‐GMP level by constitutively expressing a c‐di‐GMP synthase and it exhibited expected c‐di‐GMP‐influenced traits, such as lowered motility and increased biofilm formation. Compared to MR‐1 wild‐type strain, the high c‐di‐GMP strain had a higher Fe(III) reduction rate (21.58 vs 11.88 pM of Fe(III)/h cell) and greater expression of genes that code for the proteins involved in the Mtr pathway, including CymA, MtrA, MtrB, MtrC and OmcA. Furthermore, single‐cell Raman microspectroscopy (SCRM) revealed a great increase of c‐type cytochromes in the high c‐di‐GMP strain as compared to MR‐1 wild‐type strain. Our results reveal for the first time that the c‐di‐GMP regulation system indirectly or directly positively regulates the expression of cytochromes involved in the extracellular electron transport (EET) in S. oneidensis, which would help to understand the regulatory mechanism of c‐di‐GMP on electricity production in bacteria.

The discovery of a methanogen that can conserve energy to support growth solely from the oxidation of organic carbon coupled to the reduction of an extracellular electron acceptor expands the possible environments in which methanogens might thrive. The potential importance of c-type cytochromes for extracellular electron transfer to syntrophic bacterial partners and/or Fe(III) minerals in some Archaea was previously proposed, but these studies with Methanosarcina acetivorans provide the first genetic evidence for cytochrome-based extracellular electron transfer in Archaea. The results suggest parallels with Gram-negative bacteria, such as Shewanella and Geobacter species, in which multiheme outer-surface c-type cytochromes are an essential component for electrical communication with the extracellular environment. M. acetivorans offers an unprecedented opportunity to study mechanisms for energy conservation from the anaerobic oxidation of one-carbon organic compounds coupled to extracellular electron transfer in Archaea with implications not only for methanogens but possibly also for Archaea that anaerobically oxidize methane., Extracellular electron exchange in Methanosarcina species and closely related Archaea plays an important role in the global carbon cycle and enhances the speed and stability of anaerobic digestion by facilitating efficient syntrophic interactions. Here, we grew Methanosarcina acetivorans with methanol provided as the electron donor and the humic analogue, anthraquione-2,6-disulfonate (AQDS), provided as the electron acceptor when methane production was inhibited with bromoethanesulfonate. AQDS was reduced with simultaneous methane production in the absence of bromoethanesulfonate. Transcriptomics revealed that expression of the gene for the transmembrane, multiheme, c-type cytochrome MmcA was higher in AQDS-respiring cells than in cells performing methylotrophic methanogenesis. A strain in which the gene for MmcA was deleted failed to grow via AQDS reduction but grew with the conversion of methanol or acetate to methane, suggesting that MmcA has a specialized role as a conduit for extracellular electron transfer. Enhanced expression of genes for methanol conversion to methyl-coenzyme M and the Rnf complex suggested that methanol is oxidized to carbon dioxide in AQDS-respiring cells through a pathway that is similar to methyl-coenzyme M oxidation in methanogenic cells. However, during AQDS respiration the Rnf complex and reduced methanophenazine probably transfer electrons to MmcA, which functions as the terminal reductase for AQDS reduction. Extracellular electron transfer may enable the survival of methanogens in dynamic environments in which oxidized humic substances and Fe(III) oxides are intermittently available. The availability of tools for genetic manipulation of M. acetivorans makes it an excellent model microbe for evaluating c-type cytochrome-dependent extracellular electron transfer in Archaea.

Background Most species of Shewanella harbor two ferrochelatase paralogues for the biosynthesis of c-type cytochromes, which are crucial for their respiratory versatility. In our previous study of the Shewanella loihica PV-4 strain, we found that the disruption of hemH1 but not hemH2 resulted in a significant accumulation of extracellular protoporphyrin IX (PPIX), but it is different in Shewanella oneidensis MR-1. Hence, the function and transcriptional regulation of two ferrochelatase genes, hemH1 and hemH2, are investigated in S. oneidensis MR-1. Result In the present study, deletion of either hemH1 or hemH2 in S. oneidensis MR-1 did not lead to overproduction of extracellular protoporphyrin IX (PPIX) as previously described in the hemH1 mutants of S. loihica PV-4. Moreover, supplement of exogenous hemins made it possible to generate the hemH1 and hemH2 double mutant in MR-1, but not in PV-4. Under aerobic condition, exogenous hemins were required for the growth of MR-1ΔhemH1ΔhemH2, which also overproduced extracellular PPIX. These results suggest that heme is essential for aerobic growth of Shewanella species and MR-1 could also uptake hemin for biosynthesis of essential cytochrome(s) and respiration. Besides, the exogenous hemin mediated CymA cytochrome maturation and the cellular KatB catalase activity. Both hemH paralogues were transcribed in wild-type MR-1, and the hemH2 transcription was remarkably up-regulated in MR-1ΔhemH1 mutant to compensate for the loss of hemH1. The periplasmic glutathione peroxidase gene pgpD, located in the same operon with hemH2, and a large gene cluster coding for iron, heme (hemin) uptake systems are absent in the PV-4 genome. Conclusion Our results indicate that the genetic divergence in gene content and gene expression between these Shewanella species, accounting for the phenotypic difference described here, might be due to their speciation and adaptation to the specific habitats (iron-rich deep-sea vent versus iron-poor freshwater) in which they evolved and the generated mutants could potentially be utilized for commercial production of PPIX. Electronic supplementary material The online version of this article (10.1186/s12866-019-1549-9) contains supplementary material, which is available to authorized users.

The anaerobic corrosion of iron structures is expensive to repair and can be a safety and environmental concern. It has been known for over 100 years that the presence of anaerobic respiratory microorganisms can accelerate iron corrosion. Multiple studies have suggested that there are sulfate reducers, methanogens, and acetogens that can directly accept electrons from Fe(0) to support sulfate or carbon dioxide reduction. However, all of the strains studied can also use H2 as an electron donor for growth, which is known to be abiotically produced from Fe(0). Furthermore, no proteins definitely shown to function as extracellular electrical contacts with Fe(0) were identified. The studies described here demonstrate that direct electron transfer from Fe(0) can support anaerobic respiration. They also map out a simple genetic approach to the study of iron corrosion mechanisms in other microorganisms. A better understanding of how microorganisms promote iron corrosion is expected to lead to the development of strategies that can help reduce adverse impacts from this process., The concept that anaerobic microorganisms can directly accept electrons from Fe(0) has been controversial because direct metal-microbe electron transfer has previously only been indirectly inferred. Fe(0) oxidation was studied with Geobacter sulfurreducens strain ACL, an autotrophic strain that was previously shown to grow with electrons derived from a graphite cathode as the sole electron donor. Strain ACL grew with Fe(0) as the sole electron donor and fumarate as the electron acceptor. However, it appeared that at least a portion of the electron transfer was via H2 produced nonenzymatically from the oxidation of Fe(0) to Fe(II). H2, which accumulated in abiotic controls, was consumed during the growth of strain ACL, the cells were predominately planktonic, and genes for the uptake hydrogenase were highly expressed. Strain ACLHF was constructed to prevent growth on H2 or formate by deleting the genes for the uptake of hydrogenase and formate dehydrogenases from strain ACL. Strain ACLHF also grew with Fe(0) as the sole electron donor, but H2 accumulated in the culture, and cells heavily colonized Fe(0) surfaces with no visible planktonic growth. Transcriptomics suggested that the outer surface c-type cytochromes OmcS and OmcZ were important during growth of strain ACLHF on Fe(0). Strain ACLHF did not grow on Fe(0) if the gene for either of these cytochromes was deleted. The specific attachment of strain ACLHF to Fe(0), coupled with requirements for known extracellular electrical contacts, suggest that direct metal-microbe electron transfer is the most likely option for Fe(0) serving as an electron donor.

Geobacter bacteria are able to transfer electrons to the exterior of the cell and reduce extracellular electron acceptors including toxic/radioactive metals and electrode surfaces, with potential applications in bioremediation or electricity harvesting. The triheme c-type cytochrome PpcA from Geobacter metallireducens plays a crucial role in bridging the electron transfer from the inner to the outer membrane, ensuring an effective extracellular electron transfer. This cytochrome shares 80% identity with PpcA from Geobacter sulfurreducens, but their redox properties are markedly different, thus determining the distinctive working redox potential ranges in the two bacteria. PpcA from G. metallireducens possesses two extra aromatic amino acids (Phe-6 and Trp-45) in its hydrophobic heme core, whereas PpcA from G. sulfurreducens has a leucine and a methionine in the equivalent positions. Given the different nature of these residues in the two cytochromes, we have hypothesized that the extra aromatic amino acids could be partially responsible for the observed functional differences. In this work, we have replaced Phe-6 and Trp-45 residues by their nonaromatic counterparts in PpcA from G. sulfurreducens. Using redox titrations followed by UV–visible and NMR spectroscopy we observed that residue Trp-45 shifted the redox potential range 33% toward that of PpcA from G. sulfurreducens, whereas Phe-6 produced a negligible effect. For the first time, it is shown that the inclusion of an aromatic residue at the heme core can modulate the working redox range in abundant periplasmic proteins, paving the way to engineer bacterial strains for optimal microbial bioelectrochemical applications.

Small proteins are a new and expanding area of research. Many characterized small proteins are composed of a single hydrophobic α-helix, and the functional requirements of their limited amino acid sequence are not well understood. One hydrophobic small protein, CydX, has been shown to be a component of the cytochrome bd oxidase complex in Escherichia coli, and is required for enzyme function. To investigate small protein sequence specificity, an alanine scanning mutagenesis on the small protein CydX was conducted using mutant alleles expressed from the E. coli chromosome at the wild-type locus. The resulting mutant strains were assayed for CydX function. No single amino acid was required to maintain wild-type resistance to β-mercaptoethanol. However, substitutions of 10-amino acid blocks indicated that the N-terminus of the protein was required for wild-type CydX activity. A series of double mutants showed that multiple mutations at the N-terminus led to β-mercaptoethanol sensitivity in vivo. Triple mutants showed both in vivo and in vitro phenotypes. Together, these data provide evidence suggesting a high level of functional plasticity in CydX, in which multiple amino acids may work cooperatively to facilitate CydX function.

A microfluidic approach enables quick quantification of electrochemical activity in living bacteria., Electrons can be transported from microbes to external insoluble electron acceptors (e.g., metal oxides or electrodes in an electrochemical cell). This process is known as extracellular electron transfer (EET) and has received considerable attention due to its applications in environmental remediation and energy conversion. However, the paucity of rapid and noninvasive phenotyping techniques hinders a detailed understanding of microbial EET mechanisms. Most EET phenotyping techniques assess microorganisms based on their metabolism and growth in various conditions and/or performance in electrochemical systems, which requires large sample volumes and cumbersome experimentation. Here, we use microfluidic dielectrophoresis to show a strong correlation between bacterial EET and surface polarizability. We analyzed surface polarizabilities for wild-type strains and cytochrome-deletion mutants of two model EET microbes, Geobacter sulfurreducens and Shewanella oneidensis, and for Escherichia coli strains heterologously expressing S. oneidensis EET pathways in various growth conditions. Dielectrophoretic phenotyping is achieved with small cell culture volumes (~100 μl) in a short amount of time (1 to 2 min per strain). Our work demonstrates that cell polarizability is diminished in response to deletions of crucial outer-membrane cytochromes and enhanced due to additions of EET pathways. Results of this work hold exciting promise for rapid screening of direct EET or other cell envelope phenotypes using cell polarizability as a proxy, especially for microbes difficult to cultivate in laboratory conditions.

Iron (Fe) oxidation is one of Earth’s major biogeochemical processes, key to weathering, soil formation, water quality, and corrosion. However, our understanding of microbial contribution is limited by incomplete knowledge of microbial iron oxidation mechanisms, particularly in neutrophilic iron-oxidizers. The genomes of many, diverse iron-oxidizers encode a homolog to an outer-membrane cytochrome (Cyc2) shown to oxidize iron in two acidophiles. Phylogenetic analyses show Cyc2 sequences from neutrophiles cluster together, suggesting a common function, though this function has not been verified in these organisms. Therefore, we investigated the iron oxidase function of heterologously expressed Cyc2 from a neutrophilic iron-oxidizer Mariprofundus ferrooxydans PV-1. Cyc2PV-1 is capable of oxidizing iron, and its redox potential is 208 ± 20 mV, consistent with the ability to accept electrons from Fe2+ at neutral pH. These results support the hypothesis that Cyc2 functions as an iron oxidase in neutrophilic iron-oxidizing organisms. Sequence analysis and modeling reveal the entire Cyc2 family share a unique fused cytochrome-porin structure, with a defining consensus motif in the cytochrome region. Based on structural analyses, we predict that the monoheme cytochrome Cyc2 specifically oxidizes dissolved Fe2+, in contrast to multiheme iron oxidases, which may oxidize solid Fe(II). With our results, there is now functional validation for diverse representatives of Cyc2 sequences. We present a comprehensive Cyc2 phylogenetic tree and offer a roadmap for identifying cyc2/Cyc2 homologs and interpreting their function. The occurrence of cyc2 in many genomes beyond known iron-oxidizers presents the possibility that microbial iron oxidation may be a widespread metabolism.ImportanceIron is practically ubiquitous across Earth’s environments, central to both life and geochemical processes, which depend heavily on the redox state of iron. Although iron oxidation, or “rusting,” can occur abiotically at near neutral pH, we find neutrophilic iron-oxidizing bacteria (FeOB) are widespread, including in aquifers, sediments, hydrothermal vents, pipes, and water treatment systems. FeOB produce highly reactive Fe(III) oxyhydroxides that bind a variety of nutrients and toxins, thus these microbes are likely a controlling force in iron and other biogeochemical cycles. There has been mounting evidence that Cyc2 functions as an iron oxidase in neutrophiles, but definitive proof of its function has long eluded us. This work provides conclusive biochemical evidence of iron oxidation by Cyc2 from neutrophiles. Cyc2 is common to a wide variety of iron-oxidizers, including acidophilic and phototrophic iron-oxidizers, suggesting that this fused cytochrome-porin structure is especially well-adapted for iron oxidation.

Widely distributed outer-membrane cytochromes enable sulfur-reducing bacteria to obtain energy from solid electron donors., Bacterial reduction of oxidized sulfur species (OSS) is critical for energy production in anaerobic marine subsurfaces. In organic-poor sediments, H2 has been considered as a major energy source for bacterial respiration. We identified outer-membrane cytochromes (OMCs) that are broadly conserved in sediment OSS-respiring bacteria and enable cells to directly use electrons from insoluble minerals via extracellular electron transport. Biochemical, transcriptomic, and microscopic analyses revealed that the identified OMCs were highly expressed on the surface of cells and nanofilaments in response to electron donor limitation. This electron uptake mechanism provides sufficient but minimum energy to drive the reduction of sulfate and other OSS. These results suggest a widespread mechanism for survival of OSS-respiring bacteria via electron uptake from solid minerals in energy-poor marine sediments.

Chromosome replication in Escherichia coli is initiated by DnaA. DnaA binds ATP which is essential for formation of a DnaA-oriC nucleoprotein complex that promotes strand opening, helicase loading and replisome assembly. Following initiation, DnaAATP is converted to DnaAADP primarily by the Regulatory Inactivation of DnaA process (RIDA). In RIDA deficient cells, DnaAATP accumulates leading to uncontrolled initiation of replication and cell death by accumulation of DNA strand breaks. Mutations that suppress RIDA deficiency either dampen overinitiation or permit growth despite overinitiation. We characterize mutations of the last group that have in common that distinct metabolic routes are rewired resulting in the redirection of electron flow towards the cytochrome bd-1. We propose a model where cytochrome bd-1 lowers the formation of reactive oxygen species and hence oxidative damage to the DNA in general. This increases the processivity of replication forks generated by overinitiation to a level that sustains viability., Author Summary In most bacteria chromosome replication is initiated by the DnaA protein. In Escherichia coli, DnaA binds ATP and ADP with similar affinity but only the ATP bound form is active. An increased level of DnaAATP causes overinitiation and cell death by accumulation of DNA strand breaks. These strand breaks often result from forks encountering gapped DNA formed during repair of oxidative damage. We provide evidence that cell death in overinitiating cells can be prevented by rewiring the metabolism to favor the micro-aerobic respiratory chain with the cytochrome bd-1 as terminal oxidase. Cytochrome bd-1 is found in aerobic as well as anaerobic bacteria. Its role is to reduce O2 in micro-aerobic conditions and work as an electron sink to prevent the formation of reactive oxygen species. Our results suggest that bacteria can cope with replication stress by increasing respiration through cytochrome bd-1 to reduce the formation of reactive oxygen species, and hence oxidative damage to a level that does not interfere with replication fork progression.

For decades respiratory chain and photosystems were the main firing field of the studies devoted to mechanisms of electron transfer in proteins. The concept of conjugated lateral electron and transverse proton transport during cellular respiration and photosynthesis, which was formulated in the beginning of 1960-s, has been confirmed by thousands of experiments. However, charge transfer in recently discovered bacterial nanofilaments produced by various electrogenic bacteria is regarded currently outside of electron and proton conjugation concept. Here we report the new study of charge transfer within nanofilaments produced by Shewanella oneidensis MR-1 conducted in atmosphere of different relative humidity (RH). We utilize impedance spectroscopy and DC (direct current) transport measurements to find out the peculiarities of conductivity and Raman spectroscopy to analyze the nanofilaments' composition. Data analysis demonstrates that apparent conductivity of nanofilaments has crucial sensitivity to humidity and contains several components including one with unusual behavior which we assign to electron transport. We demonstrate that in the case of Shewanella oneidensis MR-1 charge transfer within these objects is strongly mediated by water. Basing on current data analysis of conductivity we conclude that the studied filaments of Shewanella oneidensis MR-1 are capable of hybrid (conjugated) electron and ion conductivity.

© 2015 Silveira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. "Cytochrome cd1 nitrite reductases (cd1NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd1NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd1NiR from Marinobacter hydrocarbonoclasticus (Mhcd1) depends on the presence of its physiological redox partner, cytochrome c552 (cyt c552), we show here a detailed surface enhanced resonance Raman characterization of Mhcd1 and cyt c552 attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd1 and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd1 // cyt c552 and Ag // SAM // cyt c552 // Mhcd1 constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c552 retains its native properties, while the redox potential of apparently intact Mhcd1 undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd1, reinforcing the idea that subtle and very specific interactions between Mhcd1 and cyt c552 govern efficient intermolecular electron transfer and catalytic activity of Mhcd1."

Cytochrome bd is a tri-heme (b 558, b 595, d) respiratory oxygen reductase that is found in many bacteria including pathogenic species. It couples the electron transfer from quinol to O2 with generation of an electrochemical proton gradient. We examined photolysis and subsequent recombination of CO with isolated cytochrome bd from Escherichia coli in one-electron reduced (MV) and fully reduced (R) states by microsecond time-resolved absorption spectroscopy at 532-nm excitation. Both Soret and visible band regions were examined. CO photodissociation from MV enzyme possibly causes fast (τ

Mycobacterium tuberculosis depends on aerobic respiration for growth and utilizes an aa3-type cytochrome c oxidase for terminal electron transfer. Cytochrome c maturation in bacteria requires covalent attachment of heme to apocytochrome c, which occurs outside the cytoplasmic membrane. We demonstrate that in M. tuberculosis the thioredoxin-like protein Rv3673c, which we named CcsX, is required for heme insertion in cytochrome c. Inactivation of CcsX resulted in loss of c-type heme absorbance, impaired growth and virulence of M. tuberculosis, and induced cytochrome bd oxidase. This suggests that the bioenergetically less efficient bd oxidase can compensate for deficient cytochrome c oxidase activity, highlighting the flexibility of the M. tuberculosis respiratory chain. A spontaneous mutation in the active site of vitamin K epoxide reductase (VKOR) suppressed phenotypes of the CcsX mutant and abrogated the activity of the disulfide bond-dependent alkaline phosphatase, which shows that VKOR is the major disulfide bond catalyzing protein in the periplasm of M. tuberculosis., IMPORTANCE Mycobacterium tuberculosis requires oxygen for growth; however, the biogenesis of respiratory chain components in mycobacteria has not been explored. Here, we identified a periplasmic thioredoxin, CcsX, necessary for heme insertion into cytochrome c. We investigated the consequences of disrupting cytochrome c maturation (CCM) for growth and survival of M. tuberculosis in vitro and for its pathogenesis. Appearance of a second-site suppressor mutation in the periplasmic disulfide bond catalyzing protein VKOR indicates the strong selective pressure for a functional cytochrome c oxidase. The observation that M. tuberculosis is able to partially compensate for defective CCM by upregulation of the cytochrome bd oxidase exposes a functional role of this alternative terminal oxidase under normal aerobic conditions and during pathogenesis. This suggests that targeting both oxidases simultaneously might be required to effectively disrupt respiration in M. tuberculosis.

The electrostatic interaction between plastocyanin (PC) and cytochrome f (cyt f), electron transfer partners in photosynthesis was studied using Brownian dynamics (BD) simulations. By using the software package MacroDox, which implements the BD algorithm of Northrup et al. (Northrup, S. H., J. O. Boles, and J. C. L. Reynolds. 1987. J. Phys. Chem. 91:5991–5998), we have modeled the interaction of the two proteins based on crystal structures of poplar PC and turnip cyt f at pH 7 and a variety of ionic strengths. We find that the electrostatic attraction between positively charged residues (K58, K65, K187, and R209, among others) on cyt f and negatively charged residues (E43, D44, E59, and E60, among others) on PC steers PC into a single dominant orientation with respect to cyt f, and furthermore, that the single dominant orientation that we observe is one that we had predicted in our previous work (Pearson, D. C., E. L. Gross, and E. S. David. 1996. Biophys. J. 71:64–76). This dominant orientation permits the formation of hydrophobic interactions, which are not implemented in the MacroDox algorithm. This proposed complex between PC and cyt f implicates H87, a copper ligand on PC, as the residue that accepts electrons from the heme on cyt f (and possibly through Y1 as we proposed previously). We argue for the existence of this single dominant complex on the basis of observations that the most favorable orientations of the interaction between PC and cyt f, as determined by grouping successful BD trajectories on the basis of closest contacts of charged residues, tend to overlap one another and have very close distances between the metal centers on the two proteins (copper on PC, iron on cyt f). We use this knowledge to develop a model for PC/cyt f interaction that places a reaction between the two proteins occurring when the copper-to-iron distance is between 16 and 17Å. This reaction distance gives a good estimate of the experimentally observed rate constant for PC-cyt f interaction. Analysis of BD results as a function of ionic strength predicts an interaction that happens less frequently and becomes less specific as ionic strength increases.

Cytochrome c6 is a small, soluble electron carrier between the two membrane-bound complexes cytochrome b6f and photosystem I (PSI) in oxygenic photosynthesis. We determined the solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus by NMR spectroscopy and molecular dynamics calculations based on 1586 interresidual distance and 28 dihedral angle restraints. The overall fold exhibits four alpha-helices and a small antiparallel beta-sheet in the vicinity of Met58, one of the axial heme ligands. The flat hydrophobic area in this cytochrome c6 is conserved in other c6 cytochromes and even in plastocyanin of higher plants. This docking region includes the site of electron transfer to PSI and possibly to the cytochrome b6f complex. The binding of cytochrome c6 to PSI in green algae involves interaction of a negative patch with a positive domain of PSI. This positive domain has not been inserted at the evolutionary level of cyanobacteria, but the negatively charged surface region is already present in S. elongatus cytochrome c6 and may thus have been optimized during evolution to improve the interaction with the positively charged cytochrome f. As the structure of PSI is known in S.elongatus, the reported cytochrome c6 structure can provide a basis for mutagenesis studies to delineate the mechanism of electron transfer between both.

Biogenesis of respiratory cytochromes is defined as consisting of the posttranslational processes that are necessary to assemble apoprotein, heme, and sometimes additional cofactors into mature enzyme complexes with electron transfer functions. Different biochemical reactions take place during maturation: (i) targeting of the apoprotein to or through the cytoplasmic membrane to its subcellular destination; (ii) proteolytic processing of precursor forms; (iii) assembly of subunits in the membrane and oligomerization; (iv) translocation and/or modification of heme and covalent or noncovalent binding to the protein moiety; (v) transport, processing, and incorporation of other cofactors; and (vi) folding and stabilization of the protein. These steps are discussed for the maturation of different oxidoreductase complexes, and they are arranged in a linear pathway to best account for experimental findings from studies concerning cytochrome biogenesis. The example of the best-studied case, i.e., maturation of cytochrome c, appears to consist of a pathway that requires at least nine specific genes and more general cellular functions such as protein secretion or the control of the redox state in the periplasm. Covalent attachment of heme appears to be enzyme catalyzed and takes place in the periplasm after translocation of the precursor through the membrane. The genetic characterization and the putative biochemical functions of cytochrome c-specific maturation proteins suggest that they may be organized in a membrane-bound maturase complex. Formation of the multisubunit cytochrome bc, complex and several terminal oxidases of the bo3, bd, aa3, and cbb3 types is discussed in detail, and models for linear maturation pathways are proposed wherever possible.

A minimal kinetic model of the photocycle, including both quinone (Q-6) reduction at the secondary quinone-binding site and (mammalian) cytochrome c oxidation at the cytochrome docking site of isolated reaction centers from photosynthetic purple bacteria Rhodobacter sphaeroides, was elaborated and tested by cytochrome photooxidation under strong continuous illumination. The typical rate of photochemical excitation by a laser diode at 810 nm was 2.200 s-1, and the rates of stationary turnover of the reaction center (one-half of that of cytochrome photooxidation) were 600 +/- 70 s-1 at pH 6 and 400 +/- 50 s-1 at pH 8. The rate of turnover showed strong pH dependence, indicating the contribution of different rate-limiting processes. The kinetic limitation of the photocycle was attributed to the turnover of the cytochrome c binding site (pH < 6), light intensity and quinone/quinol exchange (6 < pH < 8), and proton-coupled second electron transfer in the quinone acceptor complex (pH > 8). The analysis of the double-reciprocal plot of the rate of turnover versus light intensity has proved useful in determining the light-independent (maximum) turnover rate of the reaction center (445 +/- 50 s-1 at pH 7.8).

The binding of four anionic ligands, cyanide, fluoride, azide and formate, to cytochrome bo purified from Escherichia coli cells grown with a copper supplement (+Cu cyt.bo) is described. Membrane-bound cytochrome bo that lacks the copper component, CuB, of its active site can be prepared from cells grown under conditions where the availability of copper is limited by the presence of a CuI chelator, 2,2′-bicinchinonic acid. The ligand-binding properties of this copper-less enzyme (-Cu cyt.bo) are compared with those of +Cu cyt.bo. As judged from near-UV/visible spectroscopic changes, cyanide forms a low-spin complex with +Cu cyt.bo, whereas azide, fluoride and formate form high-spin complexes. The pH-dependences of binding suggest that for all four of these anionic ligands, both the rates of binding and the binding affinities are primarily dependent on the concentration of their protonated forms. -Cu cyt.bo, which shows less than 15% of the duroquinol oxidase activity of +Cu cyt.bo, binds cyanide, azide and fluoride, but with greatly decreased affinity (< 1/30, 1/2000 and 1/2500 respectively at pH 5.5 compared with +Cu cyt.bo). The complex of azide with -Cu cyt.bo still seems to be high-spin and azide binding to -Cu cyt.bo is still pH-dependent, although less so than azide binding to +Cu cyt.bo.

We developed a Raman spectroscopy-based approach for simultaneous study of redox changes in c-and b-type cytochromes and for a semiquantitative estimation of the amount of oxygenated myoglobin in a perfused rat heart. Excitation at 532 nm was used to obtain Raman scattering of the myocardial surface of the isolated heart at normal and hypoxic conditions. Raman spectra of the heart under normal pO2 demonstrate unique peaks attributable to reduced c-and b-type cytochromes and oxymyoglobin (oMb). The cytochrome peaks decreased in intensity upon FCCP treatment, as predicted from uncoupling mitochondrial respiration. Conversely, transient hypoxia causes the reversible increase in the intensity of peaks assigned to cytochromes c and c1, reflecting electron stacking proximal to cytochrome oxidase due to the lack of terminal electron acceptor O2. Intensities of peaks assigned to oxy- and deoxyhemoglobin were used for the semiquantitative estimation of oMb deoxygenation that was found to be of approximately 50[Formula: see text] under hypoxia conditions.

A metagenomic analysis was performed on a soil profile from a wet tundra site in northern Alaska. The goal was to link existing biogeochemical knowledge of the system with the organisms and genes responsible for the relevant metabolic pathways. We specifically investigated how the importance of iron (Fe) oxides and humic substances (HS) as terminal electron acceptors in this ecosystem is expressed genetically, and how respiratory and fermentative processes varied with soil depth into the active layer and into the upper permafrost. Overall, the metagenomes reflected a microbial community enriched in a diverse range of anaerobic pathways, with a preponderance of known Fe reducing species at all depths in the profile. The abundance of sequences associated with anaerobic metabolic processes generally increased with depth, while aerobic cytochrome c oxidases decreased. Methanogenesis genes and methanogen genomes followed the pattern of CH4 fluxes : they increased steeply with depth into the active layer, but declined somewhat over the transition zone between the lower active layer and the upper permafrost. The latter was relatively enriched in fermentative and anaerobic respiratory pathways. A survey of decaheme cytochromes (MtrA, MtrC and their homologs) revealed that this is a promising approach to identifying potential reducers of Fe(III) or HS, and indicated a possible role for Acidobacteria as Fe reducers in these soils. Methanogens appear to coexist in the same layers, though in lower abundance, with Fe reducing bacteria and other potential competitors, including acetogens. These observations provide a rich set of hypotheses for further targeted study.

Cell volume is regulated by multiple interrelated processes taking place in the cell cytoplasm and in the submembrane region. Regulation of the activity of plasma membrane ATPases is one of the important mechanisms for the maintenance of cell volume. This regulation in turn depends on the ATP production of mitochondria. It is known that mitochondrial production of ATP is heavily dependent on the dynamically changing redox status of the electron transport chain (ETC) cytochromes. At present time main methods of mitochondria studies are fluorescent microscopy with small organic dyes or genetically encoded fluorescent proteins or absorption spectroscopy and measurements of O 2 and ETC substrate consumption on isolated ETC complexes. These techniques require cell disruption or affect cell properties. Therefore, for the investigation of mitochondria-related processes of cell volume regulation it is important to develop novel, non-invasive experimental approaches. We present a non-invasive, label-free approach to study redox state of reduced cytochromes c, c1 and b of complexes II and III in mitochondria of live cardiomyocytes by means of Raman microspectroscopy. For the first time with the proposed approach we perform studies of cardiomyocytes representing different morphological and functional states. Raman mapping reveals that these cardiomyocytes differ in the amounts of reduced cytochromes c, c1 and b. The rod-shaped cardiomyocytes possess uneven distribution of reduced cytochromes c, c1 and b in cell center, and periphery. Moreover, we demonstrated the decrease in the relative amounts of reduced cytochromes c, c1 and b in the rod-shaped cardiomyocytes caused by H 2O 2-induced oxidative stress before any visible morphological changes. Results of Raman mapping and time-dependent study of reduced cytochromes of complexes II and III and cytochrome c in cardiomyocytes are in a good agreement with our fluorescence indicator studies and other published data.

The cydDC operon of Escherichia coli encodes an ATP-dependent transporter of unknown function that is required for cytochrome bd synthesis. Strains containing defects in either the cydD or cydC gene also demonstrate hypersensitivity to growth at high temperatures and the inability to exit the stationary phase at 37 degrees C. We wished to determine what is responsible for these hypersensitive phenotypes and whether they are due to a lack of the CydDC proteins or a defect of the cytochrome bd encoded by the cydAB genes. Using both K-12- and B-type strains of E. coli, we have compared the phenotypes of isogenic cydAB mutants and cydC mutants. In both K-12- and B-type backgrounds, the hypersensitive phenotypes are due to defects of cytochrome bd activity and not defects of the cydDC genes. We also found that the temperature-sensitive growth phenotypes can be suppressed by exogenous reducing agents, such as glutathione and cysteine. Strikingly, even the enzymes catalase and superoxide dismutase, when added exogenously, can correct the temperature-sensitive and stationary phase arrest phenotypes. We propose that the temperature sensitive growth phenotypes are due to a buildup of diffusible oxygen radicals brought on by the absence of cytochrome bd.

The x-ray crystallographic structure of the photosynthetic reaction center (RC) has proven critical in understanding biological electron transfer processes. By contrast, understanding of intraprotein proton transfer is easily lost in the immense richness of the details. In the RC of Rhodobacter (Rb.) sphaeroides, the secondary quinone (QB) is surrounded by amino acid residues of the L subunit and some buried water molecules, with M- and H-subunit residues also close by. The effects of site-directed mutagenesis upon RC turnover and quinone function have implicated several L-subunit residues in proton delivery to QB, although some species differences exist. In wild-type Rb. sphaeroides, Glu L212 and Asp L213 represent an inner shell of residues of particular importance in proton transfer to QB. Asp L213 is crucial for delivery of the first proton, coupled to transfer of the second electron, while Glu L212, possibly together with Asp L213, is necessary for delivery of the second proton, after the second electron transfer. We report here the first study, by site-directed mutagenesis, of the role of the H subunit in QB function. Glu H173, one of a cluster of strongly interacting residues near QB, including Asp L213, was altered to Gln. In isolated mutant RCs, the kinetics of the first electron transfer, leading to formation of the semiquinone, QB-, and the proton-linked second electron transfer, leading to the formation of fully reduced quinol, were both greatly retarded, as observed previously in the Asp L213 --> Asn mutant. However, the first electron transfer equilibrium, QA-QB QAQB-, was decreased, which is opposite to the effect of the Asp L213 --> Asn mutation. These major disruptions of events coupled to proton delivery to QB were largely reversed by the addition of azide (N3-). The results support a major role for electrostatic interactions between charged groups in determining the protonation state of certain entities, thereby controlling the rate of the second electron transfer. It is suggested that the essential electrostatic effect may be to "potentiate" proton transfer activity by raising the pK of functional entities that actually transfer protons in a coupled fashion with the second electron transfer. Candidates include buried water (H3O+) and Ser L223 (serine-OH2+), which is very close to the O5 carbonyl of the quinone.

Little in general is known about how heme proteins are assembled from their constituents in cells. The Gram-positive bacterium Enterococcus faecalis cannot synthesize heme and does not depend on it for growth. However, when supplied with heme in the growth medium the cells can synthesize two heme proteins; catalase (KatA) and cytochrome bd (CydAB). To identify novel factors important for catalase biogenesis libraries of E. faecalis gene insertion mutants were generated using two different types of transposons. The libraries of mutants were screened for clones deficient in catalase activity using a colony zymogram staining procedure. Analysis of obtained clones identified, in addition to katA (encoding the catalase enzyme protein), nine genes distributed over five different chromosomal loci. No factors with a dedicated essential role in catalase biogenesis or heme trafficking were revealed, but the results indicate the RNA degradosome (srmB, rnjA), an ABC-type oligopeptide transporter (oppBC), a two-component signal transducer (etaR), and NADH peroxidase (npr) as being important for expression of catalase activity in E. faecalis. It is demonstrated that catalase biogenesis in E. faecalis is independent of the CydABCD proteins and that a conserved proline residue in the N-terminal region of KatA is important for catalase assembly.

Light-induced charge separation in the photosynthetic reaction center results in delivery of two electrons and two protons to the terminal quinone acceptor QB. In this paper, we have used flash-induced absorbance spectroscopy to study three strains that share identical amino acid sequences in the QB binding site, all of which lack the protonatable amino acids Glu-L212 and Asp-L213. These strains are the photosynthetically incompetent site-specific mutant Glu-L212/Asp-L213-->Ala-L212/Ala-L213 and two different photocompetent derivatives that carry both alanine substitutions and an intergenic suppressor mutation located far from QB (class 3 strain, Ala-Ala + Arg-M231-->Leu; class 4 strain, Ala-Ala + Asn-M43-->Asp). At pH 8 in the double mutant, we observe a concomitant decrease of nearly 4 orders of magnitude in the rate constants of second electron and proton transfer to QB compared to the wild type. Surprisingly, these rates are increased to about the same extent in both types of suppressor strains but remain > 2 orders of magnitude smaller than those of the wild type. In the double mutant, at pH 8, the loss of Asp-L213 and Glu-L212 leads to a substantial stabilization (> or = 60 meV) of the semiquinone energy level. Both types of compensatory mutations partially restore, to nearly the same level, the original free energy difference for electron transfer from primary quinone QA to QB. The pH dependence of the electron and proton transfer processes in the double-mutant and the suppressor strains suggests that when reaction centers of the double mutant are shifted to lower pH (1.5-2 units), they function like those of the suppressor strains at physiological pH. Our data suggest that the main effect of the compensatory mutations is to partially restore the negative electrostatic environment of QB and to increase an apparent "functional" pK of the system for efficient proton transfer to the active site. This emphasizes the role of the protein in tuning the electrostatic environment of its cofactors and highlights the possible long-range electrostatic effects.

Chromium reduction by Escherichia coli ATCC 33456 quantitatively transferred hexavalent chromium, Cr(VI), to trivalent chromium, Cr(III). The reduced chromium was predominantly present in the external medium. Supernatant fluids of cell extract, obtained by centrifugation at 12,000 and 150,000 x g, showed almost the same Cr(VI) reduction activity, indicating that Cr(VI) reduction by E. coli ATCC 33456 was a largely soluble reductase activity. In studies with respiratory inhibitors, no inhibitory effects on aerobic and anaerobic Cr(VI) reduction were demonstrated by addition of cyanide, azide, and rotenone into both intact cell cultures and supernatant fluids of E. coli ATCC 33456. Although cytochromes b and d were identified in the membrane fraction of cell extracts, Cr(VI) was not reduced by the membrane fraction alone. The cytochrome difference spectra analysis also indicated that these cytochromes of the respiratory chain require the presence of the soluble Cr(VI) reductase to mediate electron transport to Cr(VI). Stimulation of Cr(VI) reduction by an uncoupler, 2,4-dinitrophenol, indicated that the respiratory-chain-linked electron transport to Cr(VI) was limited by the rate of dissipation of the proton motive force.

Shewanella putrefaciens 200 is an obligate respiratory bacterium that can utilize a variety of terminal electron acceptors, e.g., NO3-, NO2-, Fe(III), and trimethylamine N-oxide, in the absence of O2. The bacterium catalyzed the reductive transformation of tetrachloromethane (CT) under anaerobic conditions. The only identified product was trichloromethane (CF), but CF production was not stoichiometric. No dichloromethane, chloromethane, or methane was produced. A chloride mass balance indicated that fully dechlorinated products were not formed. Studies with [14C]CT suggested that a portion of the transformed CT reacted with biomass to form unidentified soluble and insoluble products. Intermediate production of a trichloromethyl radical can explain observed product distribution without significant CO2 formation. Evidence suggests that respiratory c-type cytochromes are responsible for the dehalogenation ability of S. putrefaciens 200. Previous growth under microaerobic conditions ([O2], < 2.5 microM) results in (i) a 2.6-fold increase in specific heme c content and (ii) a 2.3-fold increase in specific rates of anaerobic CT transformation. Manipulation of heme content by growth on iron-free medium or medium amended with delta-aminolevulinic acid showed that CT transformation rates increase with increases in specific heme c content. Transformation of CT is inhibited by CO. Dehalogenation studies with periplasmic, cytoplasmic, and membrane fractions indicated that only periplasmic and membrane fractions possessed dehalogenation ability. Cytochromes c were the predominant cytochromes present. Membranes were also found to contain smaller amounts of cytochrome b. Observed CT transformation patterns are consistent with a cometabolic description involving fortuitous CT reduction by reduced c-type cytochromes.

The homoacetogenic anaerobic bacterium Sporomusa sphaeroides was mutagenized with UV light. Taking advantage of the ampicillin enrichment technique and a newly developed test for the detection of heme in bacterial colonies, the cytochrome-deficient mutant strain S. sphaeroides BK824 was isolated. In contrast to the wild type, this mutant strain failed to grow on betaine, betaine plus methanol, H2 plus CO2, and methanol plus CO2. Growth on betaine plus formate, betaine plus H2, betaine plus pyruvate, methanol plus H2 and CO2, and acetoin was not impaired. All enzymes of the Wood pathway as well as hydrogenase and carbon monoxide dehydrogenase were detectable at comparable activities in both the wild type and the cytochrome-deficient mutant. Labeling experiments with [14C]methanol demonstrated the inability of S. sphaeroides BK824 to oxidize methyl groups. The role of cytochromes in electron transport steps associated with the Wood pathway enzymes and their possible role in energy conservation during autotrophic growth in acetogens are discussed.

The Escherichia coli cytochrome bd complex incorporates three haems as prosthetic groups. In the ferric form these are a predominantly high-spin chlorin (haem d), a high-spin haem b (b595) and a low-spin haem b (b558). The orientations of these three haems have been determined by e.p.r. studies on oriented multilayer preparations of cytoplasmic membrane fragments. The low-spin haem b (b558) and the high-spin haem d are oriented with their haem planes perpendicular to the membrane plane. The high-spin haem b595 is oriented with its haem plane at approx. 55 degrees to the membrane plane. A minor low-spin component, attributable to a low-spin subpopulation of the haem d, is also oriented with its haem plane perpendicular to the membrane plane.

The mechanisms by which Geobacter sulfurreducens transfers electrons through relatively thick (>50 microm) biofilms to electrodes acting as a sole electron acceptor were investigated. Biofilms of Geobacter sulfurreducens were grown either in flow-through systems with graphite anodes as the electron acceptor or on the same graphite surface, but with fumarate as the sole electron acceptor. Fumarate-grown biofilms were not immediately capable of significant current production, suggesting substantial physiological differences from current-producing biofilms. Microarray analysis revealed 13 genes in current-harvesting biofilms that had significantly higher transcript levels. The greatest increases were for pilA, the gene immediately downstream of pilA, and the genes for two outer c-type membrane cytochromes, OmcB and OmcZ. Down-regulated genes included the genes for the outer-membrane c-type cytochromes, OmcS and OmcT. Results of quantitative RT-PCR of gene transcript levels during biofilm growth were consistent with microarray results. OmcZ and the outer-surface c-type cytochrome, OmcE, were more abundant and OmcS was less abundant in current-harvesting cells. Strains in which pilA, the gene immediately downstream from pilA, omcB, omcS, omcE, or omcZ was deleted demonstrated that only deletion of pilA or omcZ severely inhibited current production and biofilm formation in current-harvesting mode. In contrast, these gene deletions had no impact on biofilm formation on graphite surfaces when fumarate served as the electron acceptor. These results suggest that biofilms grown harvesting current are specifically poised for electron transfer to electrodes and that, in addition to pili, OmcZ is a key component in electron transfer through differentiated G. sulfurreducens biofilms to electrodes.

Cytochrome d terminal oxidase mutants were isolated by using hydroxylamine mutagenesis of pNG2, a pBR322-derived plasmid containing the wild-type cyd operon. The mutagenized plasmid was transformed into a cyo cyd recA strain, and the transformants were screened for the inability to confer aerobic growth on nonfermentable carbon sources. Western blot analysis and visible-light spectroscopy were performed to characterize three independent mutants grown both aerobically and anaerobically. The mutational variants of the cytochrome d complex were stabilized under anaerobic growth conditions. All three mutations perturb the b595 and d heme components of the complex. These mutations were mapped and sequenced and are shown to be located in the N-terminal third of subunit II of the cytochrome d complex. It is proposed that the N terminus of subunit II may interact with subunit I to form an interface that binds the b595 and d heme centers.

Reactions of reduced horse spleen ferritin with horse and Saccharomyces cerevisiae ferricytochromes c, cow ferricytochrome b5, sperm-whale metmyoglobin and Pseudomonas aeruginosa ferricytochrome c-551 were investigated by u.v.-visible spectrophotometry. In all cases the reduced ferritin reduced the ferrihaemoproteins. The rate of reduction varied from less than 0.2 M-1.s-1 for metmyoglobin to 1.1 x 10(3) M-1.s-1 for horse ferricytochrome c (0.1 M-phosphate buffer, pH 7.4, at 25 degrees C). We conclude that the mechanism of ferrihaemoprotein reduction involves long-range electron transfer through the coat of ferritin and that such electron transfer is rapid enough to account for the rates of iron release observed by other workers in reductive release assays.

A genetic system has been developed for studying bacterial photosynthesis in the recently described nonsulfur purple photosynthetic bacterium Rhodospirillum centenum. Nonphotosynthetic mutants of R. centenum were obtained by enrichment for spontaneous mutations, by ethyl methanesulfonate mutagenesis coupled to penicillin selection on solid medium, and by Tn5 transposition mutagenesis with an IncP plasmid vector containing a temperature-sensitive origin of replication. In vivo and in vitro characterization of individual strains demonstrated that 38 strains contained mutations that blocked bacteriochlorophyll a biosynthesis at defined steps of the biosynthetic pathway. Collectively, these mutations were shown to block seven of eight steps of the pathway leading from protoporphyrin IX to bacteriochlorophyll a. Three mutants were isolated in which carotenoid biosynthesis was blocked early in the biosynthetic pathway; the mutants also exhibited pleiotropic effects on stability or assembly of the photosynthetic apparatus. Five mutants failed to assemble a functional reaction center complex, and seven mutants contained defects in electron transport as shown by an alteration in cytochromes. In addition, several regulatory mutants were isolated that acquired enhanced repression of bacteriochlorophyll in response to the presence of molecular oxygen. The phenotypes of these mutants are discussed in relation to those of similar mutants of Rhodobacter and other Rhodospirillum species of purple photosynthetic bacteria.

The ethanol oxidase respiratory chain of Gluconobacter suboxydan was characterized by using G. suboxydans subsp. alpha, a variant species of G. suboxydans incapable of oxidizing ethanol. The membranes of G. suboxydans subsp. alpha exhibited neither alcohol dehydrogenase, ethanol oxidase, nor glucose-ferricyanide oxidoreductase activity. Furthermore, the respiratory chain of the organism exhibited an extremely diminished amount of cytochrome c and an increased sensitivity of the respiratory activity for cyanide or azide when compared with G. suboxydans. The first-subunit quinohemoprotein and the second-subunit cytochrome c of alcohol dehydrogenase complex in the membranes of G. suboxydans subsp. alpha were shown to be reduced and deficient, respectively, by using heme-staining and immunoblotting methods. Ethanol oxidase activity, lacking in G. suboxydans subsp. alpha, was entirely restored by reconstituting alcohol dehydrogenase purified from G. suboxydans to the membranes of G. suboxydans subsp. alpha; this also led to restoration of the cyanide or azide insensitivity and the glucose-ferricyanide oxidoreductase activity in the respiratory chain without affecting other respiratory activities such as glucose and sorbitol oxidases. Ethanol oxidase activity was also reconstituted with only the second-subunit cytochrome c of the enzyme complex. The results indicate that the second-subunit cytochrome c of the alcohol dehydrogenase complex is essential in ethanol oxidase respiratory chain and may be involved in the cyanide- or azide-insensitive respiratory chain bypass of G. suboxydans.