RESUMEN
A growing number of bacteria are recognized to conduct electrons across their cell envelope, and yet molecular details of the mechanisms supporting this process remain unknown. Here, we report the atomic structure of an outer membrane spanning protein complex, MtrAB, that is representative of a protein family known to transport electrons between the interior and exterior environments of phylogenetically and metabolically diverse microorganisms. The structure is revealed as a naturally insulated biomolecular wire possessing a 10-heme cytochrome, MtrA, insulated from the membrane lipidic environment by embedding within a 26 strand ß-barrel formed by MtrB. MtrAB forms an intimate connection with an extracellular 10-heme cytochrome, MtrC, which presents its hemes across a large surface area for electrical contact with extracellular redox partners, including transition metals and electrodes.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/ultraestructura , Proteínas de la Membrana Bacteriana Externa/ultraestructura , Proteínas Bacterianas/ultraestructura , Proteínas de Unión al ARN/ultraestructura , Factores de Transcripción/ultraestructura , Transportadoras de Casetes de Unión a ATP/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Citocromos/metabolismo , Transporte de Electrón/fisiología , Electrones , Hemo/metabolismo , Complejos Multiproteicos/ultraestructura , Oxidación-Reducción , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Proteins achieve efficient energy storage and conversion through electron transfer along a series of redox cofactors. Multiheme cytochromes are notable examples. These proteins transfer electrons over distance scales of several nanometers to >10 µm and in so doing they couple cellular metabolism with extracellular redox partners including electrodes. Here, we report pump-probe spectroscopy that provides a direct measure of the intrinsic rates of heme-heme electron transfer in this fascinating class of proteins. Our study took advantage of a spectrally unique His/Met-ligated heme introduced at a defined site within the decaheme extracellular MtrC protein of Shewanella oneidensis We observed rates of heme-to-heme electron transfer on the order of 109 s-1 (3.7 to 4.3 Å edge-to-edge distance), in good agreement with predictions based on density functional and molecular dynamics calculations. These rates are among the highest reported for ground-state electron transfer in biology. Yet, some fall 2 to 3 orders of magnitude below the Moser-Dutton ruler because electron transfer at these short distances is through space and therefore associated with a higher tunneling barrier than the through-protein tunneling scenario that is usual at longer distances. Moreover, we show that the His/Met-ligated heme creates an electron sink that stabilizes the charge separated state on the 100-µs time scale. This feature could be exploited in future designs of multiheme cytochromes as components of versatile photosynthetic biohybrid assemblies.
Asunto(s)
Grupo Citocromo c/metabolismo , Citocromos/metabolismo , Electrones , Hemo/metabolismo , Histidina/metabolismo , Metionina/metabolismo , Shewanella/metabolismo , Grupo Citocromo c/química , Citocromos/química , Transporte de Electrón , Hemo/química , Histidina/química , Metionina/química , Simulación de Dinámica Molecular , Nanocables , Oxidación-ReducciónRESUMEN
A growing number of bacterial species are known to move electrons across their cell envelopes. Naturally this occurs in support of energy conservation and carbon-fixation. For biotechnology it allows electron exchange between bacteria and electrodes in microbial fuel cells and during microbial electrosynthesis. In this context Rhodopseudomonas palustris TIE-1 is of much interest. These bacteria respond to light by taking electrons from their external environment, including electrodes, to drive CO2-fixation. The PioA cytochrome, that spans the bacterial outer membrane, is essential for this electron transfer and yet little is known about its structure and electron transfer properties. Here we reveal the ten c-type hemes of PioA are redox active across the window +250 to -400 mV versus Standard Hydrogen Electrode and that the hemes with most positive reduction potentials have His/Met and His/H2O ligation. These chemical and redox properties distinguish PioA from the more widely studied family of MtrA outer membrane decaheme cytochromes with ten His/His ligated hemes. We predict a structure for PioA in which the hemes form a chain spanning the longest dimension of the protein, from Heme 1 to Heme 10. Hemes 2, 3 and 7 are identified as those most likely to have His/Met and/or His/H2O ligation. Sequence analysis suggests His/Met ligation of Heme 2 and/or 7 is a defining feature of decaheme PioA homologs from over 30 different bacterial genera. His/Met ligation of Heme 3 appears to be less common and primarily associated with PioA homologs from purple non-sulphur bacteria belonging to the alphaproteobacteria class.
Asunto(s)
Citocromos/química , Citocromos/metabolismo , Hemo/química , Rhodopseudomonas/fisiología , Membrana Externa Bacteriana/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dióxido de Carbono/metabolismo , Técnicas Electroquímicas , Transporte de Electrón , Modelos Moleculares , Fotosíntesis , Conformación ProteicaRESUMEN
Many subsurface microorganisms couple their metabolism to the reduction or oxidation of extracellular substrates. For example, anaerobic mineral-respiring bacteria can use external metal oxides as terminal electron acceptors during respiration. Porin-cytochrome complexes facilitate the movement of electrons generated through intracellular catabolic processes across the bacterial outer membrane to these terminal electron acceptors. In the mineral-reducing model bacterium Shewanella oneidensis MR-1, this complex is composed of two decaheme cytochromes (MtrA and MtrC) and an outer-membrane ß-barrel (MtrB). However, the structures and mechanisms by which porin-cytochrome complexes transfer electrons are unknown. Here, we used small-angle neutron scattering (SANS) to study the molecular structure of the transmembrane complexes MtrAB and MtrCAB. Ab initio modeling of the scattering data yielded a molecular envelope with dimensions of â¼105 × 60 × 35 Å for MtrAB and â¼170 × 60 × 45 Å for MtrCAB. The shapes of these molecular envelopes suggested that MtrC interacts with the surface of MtrAB, extending â¼70 Å from the membrane surface and allowing the terminal hemes to interact with both MtrAB and an extracellular acceptor. The data also reveal that MtrA fully extends through the length of MtrB, with â¼30 Å being exposed into the periplasm. Proteoliposome models containing membrane-associated MtrCAB and internalized small tetraheme cytochrome (STC) indicate that MtrCAB could reduce Fe(III) citrate with STC as an electron donor, disclosing a direct interaction between MtrCAB and STC. Taken together, both structural and proteoliposome experiments support porin-cytochrome-mediated electron transfer via periplasmic cytochromes such as STC.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Proteínas de la Membrana Bacteriana Externa/química , Proteínas Bacterianas/química , Membrana Celular/metabolismo , Grupo Citocromo c/química , Electrones , Metales/química , Periplasma/metabolismo , Shewanella/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Respiración de la Célula , Cristalografía por Rayos X , Grupo Citocromo c/metabolismo , Transporte de Electrón , Metales/metabolismo , Oxidación-ReducciónRESUMEN
Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits. The cell surface cytochromes can potentially play multiple roles in mediating electron transfer directly to insoluble electron sinks, catalyzing electron exchange with flavin electron shuttles or participating in extracellular intercytochrome electron exchange along "nanowire" appendages. We present a 3.2-Å crystal structure of one of these decaheme cytochromes, MtrF, that allows the spatial organization of the 10 hemes to be visualized for the first time. The hemes are organized across four domains in a unique crossed conformation, in which a staggered 65-Å octaheme chain transects the length of the protein and is bisected by a planar 45-Å tetraheme chain that connects two extended Greek key split ß-barrel domains. The structure provides molecular insight into how reduction of insoluble substrate (e.g., minerals), soluble substrates (e.g., flavins), and cytochrome redox partners might be possible in tandem at different termini of a trifurcated electron transport chain on the cell surface.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Grupo Citocromo c/química , Citocromos/química , Hemo/química , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Sitios de Unión/genética , Cristalografía por Rayos X , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Grupo Citocromo c/genética , Grupo Citocromo c/metabolismo , Citocromos/genética , Citocromos/metabolismo , Disulfuros/química , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Mononucleótido de Flavina/química , Mononucleótido de Flavina/metabolismo , Mononucleótido de Flavina/farmacología , Hemo/metabolismo , Hierro/química , Hierro/metabolismo , Hierro/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción/efectos de los fármacos , Potenciometría , Unión Proteica , Estructura Terciaria de Proteína , Shewanella/genética , Shewanella/metabolismoRESUMEN
Many species of bacteria can couple anaerobic growth to the respiratory reduction of insoluble minerals containing Fe(III) or Mn(III/IV). It has been suggested that in Shewanella species electrons cross the outer membrane to extracellular substrates via 'porin-cytochrome' electron transport modules. The molecular structure of an outer-membrane extracellular-facing deca-haem terminus for such a module has recently been resolved. It is debated how, once outside the cells, electrons are transferred from outer-membrane cytochromes to insoluble electron sinks. This may occur directly or by assemblies of cytochromes, perhaps functioning as 'nanowires', or via electron shuttles. Here we review recent work in this field and explore whether it allows for unification of the electron transport mechanisms supporting extracellular mineral respiration in Shewanella that may extend into other genera of Gram-negative bacteria.
Asunto(s)
Citocromos/metabolismo , Transporte de Electrón , Minerales/metabolismo , Porinas/metabolismo , Shewanella/fisiología , Anaerobiosis , Modelos Biológicos , Oxidación-Reducción , Shewanella/crecimiento & desarrollo , Shewanella/metabolismoRESUMEN
Electroactive bacteria combine the oxidation of carbon substrates with an extracellular electron transfer (EET) process that discharges electrons to an electron acceptor outside the cell. This process involves electron transfer through consecutive redox proteins that efficiently connect the inner membrane to the cell exterior. In this study, we isolated and characterized the quinone-interacting membrane cytochrome c ImcH from Geobacter sulfurreducens, which is involved in the EET process to high redox potential acceptors. Spectroscopic and electrochemical studies show that ImcH hemes have low midpoint redox potentials, ranging from -150 to -358 mV, and connect the oxidation of the quinol-pool to EET, transferring electrons to the highly abundant periplasmic cytochrome PpcA with higher affinity than to its homologues. Despite the larger number of hemes and transmembrane helices, the ImcH structural model has similarities with the NapC/NirT/NrfH superfamily, namely the presence of a quinone-binding site on the P-side of the membrane. In addition, the first heme, likely involved on the quinol oxidation, has apparently an unusual His/Gln coordination. Our work suggests that ImcH is electroneutral and transfers electrons and protons to the same side of the membrane, contributing to the maintenance of a proton motive force and playing a central role in recycling the menaquinone pool.
Asunto(s)
Electrones , Geobacter , Hidroquinonas/metabolismo , Geobacter/metabolismo , Proteínas Bacterianas/química , Transporte de Electrón , Oxidación-Reducción , Citocromos c/metabolismo , Quinonas/metabolismoRESUMEN
Many bacteria of the genus Shewanella are facultative anaerobes able to reduce a broad range of soluble and insoluble substrates, including Fe(III) mineral oxides. Under anoxic conditions, the bacterium Shewanella oneidensis MR-1 uses a porin-cytochrome complex (Mtr) to mediate extracellular electron transfer (EET) across the outer membrane to extracellular substrates. However, it is unclear how EET prevents generating harmful reactive oxygen species (ROS) when exposed to oxic environments. The Mtr complex is expressed under anoxic and oxygen-limited conditions and contains an extracellular MtrC subunit. This has a conserved CX8C motif that inhibits aerobic growth when removed. This inhibition is caused by an increase in ROS that kills the majority of S. oneidensis cells in culture. To better understand this effect, soluble MtrC isoforms with modified CX8C were isolated. These isoforms produced increased concentrations of H2O2 in the presence of flavin mononucleotide (FMN) and greatly increased the affinity between MtrC and FMN. X-ray crystallography revealed that the molecular structure of MtrC isoforms was largely unchanged, while small-angle X-ray scattering suggested that a change in flexibility was responsible for controlling FMN binding. Together, these results reveal that FMN reduction in S. oneidensis MR-1 is controlled by the redox-active disulfide on the cytochrome surface. In the presence of oxygen, the disulfide forms, lowering the affinity for FMN and decreasing the rate of peroxide formation. This cysteine pair consequently allows the cell to respond to changes in oxygen level and survive in a rapidly transitioning environment. IMPORTANCE Bacteria that live at the oxic/anoxic interface have to rapidly adapt to changes in oxygen levels within their environment. The facultative anaerobe Shewanella oneidensis MR-1 can use EET to respire in the absence of oxygen, but on exposure to oxygen, EET could directly reduce extracellular oxygen and generate harmful reactive oxygen species that damage the bacterium. By modifying an extracellular cytochrome called MtrC, we show how preventing a redox-active disulfide from forming causes the production of cytotoxic concentrations of peroxide. The disulfide affects the affinity of MtrC for the redox-active flavin mononucleotide, which is part of the EET pathway. Our results demonstrate how a cysteine pair exposed on the surface controls the path of electron transfer, allowing facultative anaerobic bacteria to rapidly adapt to changes in oxygen concentration at the oxic/anoxic interface.
Asunto(s)
Cisteína , Shewanella , Especies Reactivas de Oxígeno/metabolismo , Cisteína/metabolismo , Compuestos Férricos/metabolismo , Mononucleótido de Flavina/metabolismo , Peróxido de Hidrógeno/metabolismo , Oxidación-Reducción , Citocromos/metabolismo , Transporte de Electrón , Shewanella/genética , Shewanella/metabolismo , Flavinas/metabolismo , Oxígeno/metabolismo , Disulfuros/metabolismoRESUMEN
The outer-membrane decahaem cytochrome MtrC is part of the transmembrane MtrCAB complex required for mineral respiration by Shewanella oneidensis. MtrC has significant sequence similarity to the paralogous decahaem cytochrome MtrF, which has been structurally solved through X-ray crystallography. This now allows for homology-based models of MtrC to be generated. The structure of these MtrC homology models contain ten bis-histidine-co-ordinated c-type haems arranged in a staggered cross through a four-domain structure. This model is consistent with current spectroscopic data and shows that the areas around haem 5 and haem 10, at the termini of an octahaem chain, are likely to have functions similar to those of the corresponding haems in MtrF. The electrostatic surfaces around haem 7, close to the ß-barrels, are different in MtrF and MtrC, indicating that these haems may have different potentials and interact with substrates differently.
Asunto(s)
Grupo Citocromo c/química , Shewanella , Secuencia de Aminoácidos , Sitios de Unión , Hemo/química , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Homología Estructural de ProteínaRESUMEN
The free energy profile for electron flow through the bacterial decahaem cytochrome MtrF has been computed using thermodynamic integration and classical molecular dynamics. The extensive calculations on two versions of the structure help to validate the method and results, because differences in the profiles can be related to differences in the charged amino acids local to specific haem groups. First estimates of reorganization free energies λ yield a range consistent with expectations for partially solvent-exposed cofactors, and reveal an activation energy range surmountable for electron flow. Future work will aim at increasing the accuracy of λ with polarizable forcefield dynamics and quantum chemical energy gap calculations, as well as quantum chemical computation of electronic coupling matrix elements.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Citocromos/química , Shewanella/metabolismo , Transporte de Electrón , Hemo/química , Simulación de Dinámica Molecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , TermodinámicaRESUMEN
Many species of the bacterial Shewanella genus are notable for their ability to respire in anoxic environments utilizing insoluble minerals of Fe(III) and Mn(IV) as extracellular electron acceptors. In Shewanella oneidensis, the process is dependent on the decahaem electron-transport proteins that lie at the extracellular face of the outer membrane where they can contact the insoluble mineral substrates. These extracellular proteins are charged with electrons provided by an inter-membrane electron-transfer pathway that links the extracellular face of the outer membrane with the inner cytoplasmic membrane and thereby intracellular electron sources. In the present paper, we consider the common structural features of two of these outer-membrane decahaem cytochromes, MtrC and MtrF, and bring this together with biochemical, spectroscopic and voltammetric data to identify common and distinct properties of these prototypical members of different clades of the outer-membrane decahaem cytochrome superfamily.
Asunto(s)
Espacio Extracelular/metabolismo , Hierro/metabolismo , Minerales/metabolismo , Shewanella/metabolismo , Aerobiosis , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Shewanella/genéticaRESUMEN
DNA topoisomerases are enzymes that control DNA topology and are vital targets for antimicrobial and anticancer drugs. Here we present a mass spectrometry study of complexes formed between the A subunit of the topoisomerase DNA gyrase and the bifunctional inhibitor simocyclinone D8 (SD8), an antibiotic isolated from Streptomyces. These studies show that, in an alternative mode of interaction to that found by X-ray crystallography, each subunit binds a single bifunctional inhibitor with separate binding pockets for the two ends of SD8. The gyrase subunits form constitutive dimers, and fractional occupancies of inhibitor-bound states show that there is strong allosteric cooperativity in the binding of two bifunctional ligands to the dimer. We show that the mass spectrometry data can be fitted to a general model of cooperative binding via an extension of the "tight-binding" approach, providing a rigorous determination of the dissociation constants and degree of cooperativity. This general approach will be applicable to other systems with multiple binding sites and highlights mass spectrometry's role as a powerful emerging tool for unraveling the complexities of biomolecular interactions.
Asunto(s)
Antibacterianos/química , Girasa de ADN/química , Regulación Alostérica , Cumarinas/química , Glicósidos/química , Espectrometría de Masas , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Subunidades de Proteína/químicaRESUMEN
Shewanella oneidensis exchanges electrons between cellular metabolism and external redox partners in a process that attracts much attention for production of green electricity (microbial fuel cells) and chemicals (microbial electrosynthesis). A critical component of this pathway is the outer membrane spanning MTR complex, a biomolecular wire formed of the MtrA, MtrB, and MtrC proteins. MtrA and MtrC are decaheme cytochromes that form a chain of close-packed hemes to define an electron transfer pathway of 185 Å. MtrA is wrapped inside MtrB for solubility across the outer membrane lipid bilayer; MtrC sits outside the cell for electron exchange with external redox partners. Here, we demonstrate tight and spontaneous in vitro association of MtrAB with separately purified MtrC. The resulting complex is comparable with the MTR complex naturally assembled by Shewanella in terms of both its structure and rates of electron transfer across a lipid bilayer. Our findings reveal the potential for building bespoke electron conduits where MtrAB combines with chemically modified MtrC, in this case, labeled with a Ru-dye that enables light-triggered electron injection into the MtrC heme chain.
RESUMEN
Heme containing proteins are involved in a broad range of cellular functions, from oxygen sensing and transport to catalyzing oxidoreductive reactions. The two major types of cytochrome (b-type and c-type) only differ in their mechanism of heme attachment, but this has major implications for their cellular roles in both localization and mechanism. The b-type cytochromes are commonly cytoplasmic, or are within the cytoplasmic membrane, while c-type cytochromes are always found outside of the cytoplasm. The mechanism of heme attachment allows for complex c-type multiheme complexes, having the capacity to hold multiple electrons, to be assembled. These are increasingly being identified as secreted into the extracellular environment. For organisms that respire using extracellular substrates, these large multiheme cytochromes allow for electron transfer networks from the cytoplasmic membrane to the cell exterior for the reduction of extracellular electron acceptors. In this review the structures and functions of these networks and the mechanisms by which electrons are transferred to extracellular substrates is described.
Asunto(s)
Bacterias/metabolismo , Citocromos/metabolismo , Anaerobiosis , Bacterias/química , Citocromos/química , Transporte de Electrón , ElectronesRESUMEN
Microbial nanowires are fascinating biological structures that allow bacteria to transport electrons over micrometers for reduction of extracellular substrates. It was recently established that the nanowires of both Shewanella and Geobacter are made of multi-heme proteins; but, while Shewanella employs the 20-heme protein complex MtrCAB, Geobacter uses a redox polymer made of the hexa-heme protein OmcS, begging the question as to which protein architecture is more efficient in terms of long-range electron transfer. Using a multiscale computational approach we find that OmcS supports electron flows about an order of magnitude higher than MtrCAB due to larger heme-heme electronic couplings and better insulation of hemes from the solvent. We show that heme side chains are an essential structural element in both protein complexes, accelerating rate-limiting electron tunnelling steps up to 1000-fold. Our results imply that the alternating stacked/T-shaped heme arrangement present in both protein complexes may be an evolutionarily convergent design principle permitting efficient electron transfer over very long distances.
Asunto(s)
Proteínas Bacterianas/química , Hemo/química , Hemoproteínas/química , Transporte de Electrón , Geobacter/química , Nanocables/química , Oxidación-Reducción , Conformación Proteica , Shewanella/química , Solventes/química , Relación Estructura-ActividadRESUMEN
Crystals of a complex formed between the 59 kDa N-terminal fragment of the Escherichia coli DNA gyrase A subunit (also known as the breakage-reunion domain) and the antibiotic simocyclinone D8 were grown by vapour diffusion. The complex crystallized with I-centred orthorhombic symmetry and X-ray data were recorded to a resolution of 2.75 A from a single crystal at the synchrotron. DNA gyrase is an essential bacterial enzyme and thus represents an attractive target for drug development.
Asunto(s)
Daño del ADN , Girasa de ADN/química , Escherichia coli/enzimología , Cumarinas/química , Cristalización , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Glicósidos/química , Conformación MolecularRESUMEN
Certain bacterial species have a natural ability to exchange electrons with extracellular redox partners. This behavior allows coupling of catalytic transformations inside bacteria to complementary redox transformations of catalysts and electrodes outside the cell. Electricity generation can be coupled to waste-water remediation. Industrially relevant oxidation reactions can proceed exclusively when electrons are released to anodes. Reduced products such as fuels can be generated when electrons are provided from (photo)cathodes. Rational development of these opportunities and inspiration for novel technologies is underpinned by resolution at the molecular level of pathways supporting electron exchange across bacterial cell envelopes. This chapter describes methods for purification, engineering, and in vitro characterization of proteins providing the primary route for electron transport across the outer membrane lipid bilayer of Shewanella oneidensis MR-1, a well-described electrogenic bacterium and chassis organism for related biotechnologies.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Transporte de Electrón/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Oxidación-Reducción , Shewanella/enzimologíaRESUMEN
The autotrophic Sideroxydans lithotrophicus ES-1 can grow by coupling the oxidation of ferrous iron to the reduction of oxygen. Soluble ferrous iron is oxidized at the surface of the cell by an MtoAB porin-cytochrome complex that functions as an electron conduit through the outer membrane. Electrons are then transported to the cytoplasmic membrane where they are used to generate proton motive force (PMF) (for ATP synthesis) and NADH for autotrophic processes such as carbon fixation. As part of the mtoAB gene cluster, S. lithotrophicus also contains the gene mtoD that is proposed to encode a cytochrome c protein. We isolated mtoD from a Shewanella oneidensis expression system where the mtoD gene was expressed on a pBAD plasmid vector. Biochemical, biophysical, and crystallographic characterization of the purified MtoD revealed it as an 11 kDa monomeric protein containing a single heme. Sequence and structural alignment indicated that MtoD belonged to the class-1 cytochrome c family and had a similar fold to ferricytochrome c552 family, however the MtoD heme is bis-histidine coordinated and is substantially more exposed than the hemes of other family members. The reduction potential of the MtoD heme at pH 7 was +155 mV vs. Standard Hydrogen Electrode, which is approximately 100 mV lower than that of mitochondrial cytochrome c. Consideration of the properties of MtoD in the context of the potential respiratory partners identified from the genome suggests that MtoD could associate to multiple electron transfer partners as the primary periplasmic electron shuttle.
RESUMEN
Extracellular microbe-mineral electron transfer is a major driving force for the oxidation of organic carbon in many subsurface environments. Extracellular multi-heme cytochromes of the Shewenella genus play a major role in this process but the mechanism of electron exchange at the interface between cytochrome and acceptor is widely debated. The 1.8 Å x-ray crystal structure of the decaheme MtrC revealed a highly conserved CX8C disulfide that, when substituted for AX8A, severely compromised the ability of S. oneidensis to grow under aerobic conditions. Reductive cleavage of the disulfide in the presence of flavin mononucleotide (FMN) resulted in the reversible formation of a stable flavocytochrome. Similar results were also observed with other decaheme cytochromes, OmcA, MtrF and UndA. The data suggest that these decaheme cytochromes can transition between highly reactive flavocytochromes or less reactive cytochromes, and that this transition is controlled by a redox active disulfide that responds to the presence of oxygen.