Functional Differentiation of Antiporter-Like Polypeptides in Complex I; a Site-Directed Mutagenesis Study of Residues Conserved in MrpA and NuoL but Not in MrpD, NuoM, and NuoN.

It has long been known that the three largest subunits in the membrane domain (NuoL, NuoM and NuoN) of complex I are homologous to each other, as well as to two subunits (MrpA and MrpD) from a Na+/H+ antiporter, Mrp. MrpA and NuoL are more similar to each other and the same is true for MrpD and NuoN. This suggests a functional differentiation which was proven experimentally in a deletion strain model system, where NuoL could restore the loss of MrpA, but not that of MrpD and vice versa. The simplest explanation for these observations was that the MrpA and MrpD proteins are not antiporters, but rather single subunit ion channels that together form an antiporter. In this work our focus was on a set of amino acid residues in helix VIII, which are only conserved in NuoL and MrpA (but not in any of the other antiporter-like subunits.) and to compare their effect on the function of these two proteins. By combining complementation studies in B. subtilis and 23Na-NMR, response of mutants to high sodium levels were tested. All of the mutants were able to cope with high salt levels; however, all but one mutation (M258I/M225I) showed differences in the efficiency of cell growth and sodium efflux. Our findings showed that, although very similar in sequence, NuoL and MrpA seem to differ on the functional level. Nonetheless the studied mutations gave rise to interesting phenotypes which are of interest in complex I research.

Photo-electrochemical communication between cyanobacteria (Leptolyngbia sp.) and osmium redox polymer modified electrodes.

Photosynthetic microbial fuel cells (PMFCs) are an emerging technology for renewable solar energy conversion. Major efforts have been made to explore the electrogenic activity of cyanobacteria, mostly using practically unsustainable reagents. Here we report on photocurrent generation (≈8.64 µA cm(-2)) from cyanobacteria immobilized on electrodes modified with an efficient electron mediator, an Os(2+/3+) redox polymer. Upon addition of ferricyanide to the electrolyte, cyanobacteria generate the maximum current density of ≈48.2 µA cm(-2).

Functional role of the MrpA- and MrpD-homologous protein subunits in enzyme complexes evolutionary related to respiratory chain complex I.

NADH:quinone oxidoreductase or complex I is a large membrane bound enzyme complex that has evolved from the combination of smaller functional building blocks. Intermediate size enzyme complexes exist in nature that comprise some, but not all of the protein subunits in full size 14-subunit complex I. The membrane spanning complex I subunits NuoL, NuoM and NuoN are homologous to each other and to two proteins from one particular class of Na(+)/H(+) antiporters, denoted MrpA and MrpD. In complex I, these ion transporter protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Using a model system, consisting of Bacillus subtilis MrpA and MrpD deletion strains and a low copy expression plasmid, it was recently demonstrated that NuoN can rescue the strain deleted for MrpD but not that deleted for MrpA, whereas the opposite tendency was seen for NuoL. This demonstrated that the MrpA-type and MrpD-type proteins have unique functional specializations. In this work, the corresponding antiporter-like protein subunits from the smaller enzymes evolutionarily related to complex I were tested in the same model system. The subunits from 11-subunit complex I from Bacillus cereus behaved essentially as those from full size complex I, corroborating that this enzyme should be regarded as a bona fide complex I. The hydrogenase-3 and hydrogenase-4 antiporter-like proteins on the other hand, could substitute equally well for MrpA or MrpD at pH7.4, suggesting that these enzymes have intermediate forms of the antiporter-like proteins, which seemingly lack the functional specificity.

The Na+ transport in gram-positive bacteria defect in the Mrp antiporter complex measured with 23Na nuclear magnetic resonance.

(23)Na nuclear magnetic resonance (NMR) has previously been used to monitor Na(+) translocation across membranes in gram-negative bacteria and in various other organelles and liposomes using a membrane-impermeable shift reagent to resolve the signals resulting from internal and external Na(+). In this work, the (23)Na NMR method was adapted for measurements of internal Na(+) concentration in the gram-positive bacterium Bacillus subtilis, with the aim of assessing the Na(+) translocation activity of the Mrp (multiple resistance and pH) antiporter complex, a member of the cation proton antiporter-3 (CPA-3) family. The sodium-sensitive growth phenotype observed in a B. subtilis strain with the gene encoding MrpA deleted could indeed be correlated to the inability of this strain to maintain a lower internal Na(+) concentration than an external one.

Structure and function of the C-terminal domain of MrpA in the Bacillus subtilis Mrp-antiporter complex--the evolutionary progenitor of the long horizontal helix in complex I.

MrpA and MrpD are homologous to NuoL, NuoM and NuoN in complex I over the first 14 transmembrane helices. In this work, the C-terminal domain of MrpA, outside this conserved area, was investigated. The transmembrane orientation was found to correspond to that of NuoJ in complex I. We have previously demonstrated that the subunit NuoK is homologous to MrpC. The function of the MrpA C-terminus was tested by expression in a previously used Bacillus subtilis model system. At neutral pH, the truncated MrpA still worked, but at pH 8.4, where Mrp-complex formation is needed for function, the C-terminal domain of MrpA was absolutely required.

Electrospun carbon nanofibers from polyacrylonitrile blended with activated or graphitized carbonaceous materials for improving anodic bioelectrocatalysis.

The electrospun carbon nanofibers obtained from polyacrylonitrile (PAN) and PAN blends with either activated carbon (PAN-AC) or graphite (PAN-GR) were tested as anodes using Shewanella oneidensis MR-1. Extensive physico-chemical and electrochemical characterization confirmed their formation, their fibrous and porous nature, and their suitability as electrodes. N2 adsorption measurements revealed high specific surface area (229.8, 415.8 and 485.2m(2) g(-1)) and porosity (0.142, 0.202 and 0.239cm(3)g(-1)) for PAN, PAN-AC and PAN-GR, respectively. The chronoamperometric measurements showed a considerable decrease in start-up time and more than a 10-fold increase in the generation of current with these electrodes (115, 139 and 155µAcm(-2) for PAN, PAN-AC and PAN-GR, respectively) compared to the graphite electrode (11.5µAcm(-2)). These results indicate that the bioelectrocatalysis benefits from the blending of PAN with activated or graphitized carbonaceous materials, presumably due to the increased specific surface area, total pore volume and modification of the carbon microstructure.

Electrochemical communication between heterotrophically grown Rhodobacter capsulatus with electrodes mediated by an osmium redox polymer.

The metabolically versatile purple bacteria Rhodobacter capsulatus was investigated to check its possible applicability in biofuel cells and electrochemical microbial biosensors. The wild type strain ATCC 17015 and mutant strain 37b4 lacking the lipopolysaccharide capsule was compared for their ability to communicate with electrodes modified with an osmium redox polymer. In this work, aerobic heterotrophically grown R. capsulatus were used to screen for efficient cell-electrode communication for later implementation using photoheterotrophically grown bacteria. The bacterial cells embedded in the osmium polymer matrix demonstrated efficient electrical "wiring" with the electrodes and were able to generate a noticeable current with succinate as substrate. Interestingly, at 2mM succinate the wild type strain showed much better bioelectrocatalytic current generation (4.25 µA/cm(2)) than the strain lacking capsule (1.55 µA/cm(2)). The wild type strain also exhibited a stable current response for longer time, demonstrating that the bacterial lipopolysaccharide in fact enhances the stability of the polymer matrix layer of the modified electrode. Control experiments with R. capsulatus without any mediator did not show any current irrespective of the capsule presence. This demonstrates that development of photosensors and other light driven bioelectrochemical devices could be feasible using R. capsulatus and will be at focus for future studies.

Electrochemical communication between microbial cells and electrodes via osmium redox systems.

Electrochemical communication between micro-organisms and electrodes is the integral and fundamental part of BESs (bioelectrochemical systems). The immobilization of bacterial cells on the electrode and ensuring efficient electron transfer to the electrode via a mediator are decisive features of mediated electrochemical biosensors. Notably, mediator-based systems are essential to extract electrons from the non-exoelectrogens, a major group of microbes in Nature. The advantage of using polymeric mediators over diffusible mediators led to the design of osmium redox polymers. Their successful use in enzyme-based biosensors and BFCs (biofuel cells) paved the way for exploring their use in microbial BESs. The present mini-review focuses on osmium-bound redox systems used to date in microbial BESs and their role in shuttling electrons from viable microbial cells to electrodes.

Improved microbial electrocatalysis with osmium polymer modified electrodes.

Using the well-known exoelectrogen Shewanella oneidensis MR-1, an osmium redox polymer modified anode exhibited ca. 4-fold increase in current generation. Additionally, a significant decrease in the start-up time for electrocatalysis was observed. The findings suggest that the inherent extracellular electron transfer capabilities of electrogens coupled with such polymers could enhance electrocatalysis.

Revised transmembrane orientation of the NADH:quinone oxidoreductase subunit NuoA.

NuoA is a small membrane spanning subunit of respiratory chain NADH:quinone oxidoreductase (complex I). Unlike the other complex I core protein subunits, the NuoA protein has no known homologue in other enzyme systems. The transmembrane orientation of NuoA cannot be unambiguously predicted, due to the small size of the polypeptide and the varying distribution of charged amino acid residues in NuoA from different organisms. Novel analyses of NuoA from Escherichia coli complex I expressed as fusion proteins to cytochrome c and to alkaline phosphatase demonstrated that the c-terminal end of the polypeptide is localized in the bacterial cytoplasm, in contrast to what was previously reported for the homologous NQO7 subunit from Paracoccus denitrificans complex I.

The evolution of respiratory chain complex I from a smaller last common ancestor consisting of 11 protein subunits.

The NADH:quinone oxidoreductase (complex I) has evolved from a combination of smaller functional building blocks. Chloroplasts and cyanobacteria contain a complex I-like enzyme having only 11 subunits. This enzyme lacks the N-module which harbors the NADH binding site and the flavin and iron-sulfur cluster prosthetic groups. A complex I-homologous enzyme found in some archaea contains an F(420) dehydrogenase subunit denoted as FpoF rather than the N-module. In the present study, all currently available whole genome sequences were used to survey the occurrence of the different types of complex I in the different kingdoms of life. Notably, the 11-subunit version of complex I was found to be widely distributed, both in the archaeal and in the eubacterial kingdoms, whereas the 14-subunit classical complex I was found only in certain eubacterial phyla. The FpoF-containing complex I was present in Euryarchaeota but not in Crenarchaeota, which contained the 11-subunit complex I. The 11-subunit enzymes showed a primary sequence variability as great or greater than the full-size 14-subunit complex I, but differed distinctly from the membrane-bound hydrogenases. We conclude that this type of compact 11-subunit complex I is ancestral to all present-day complex I enzymes. No designated partner protein, acting as an electron delivery device, could be found for the compact version of complex I. We propose that the primordial complex I, and many of the present-day 11-subunit versions of it, operate without a designated partner protein but are capable of interaction with several different electron donor or acceptor proteins.

Homologous protein subunits from Escherichia coli NADH:quinone oxidoreductase can functionally replace MrpA and MrpD in Bacillus subtilis.

The complex I subunits NuoL, NuoM and NuoN are homologous to two proteins, MrpA and MrpD, from one particular class of Na+/H+ antiporters. In many bacteria MrpA and MrpD are encoded by an operon comprising 6-7 conserved genes. In complex I these protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Deletion of either mrpA or mrpD from the Bacillus subtilis chromosome resulted in a Na+ and pH sensitive growth phenotype. The deletion strains could be complemented in trans by their respective Mrp protein, but expression of MrpA in the B. subtilis ΔmrpD strain and vice versa did not improve growth at pH 7.4. This corroborates that the two proteins have unique specific functions. Under the same conditions NuoL could rescue B. subtilis ΔmrpA, but improved the growth of B. subtilis ΔmrpD only slightly. NuoN could restore the wild type properties of B. subtilis ΔmrpD, but had no effect on the ΔmrpA strain. Expression of NuoM did not result in any growth improvement under these conditions. This reveals that the complex I subunits NuoL, NuoM and NuoN also demonstrate functional specializations. The simplest explanation that accounts for all previous and current observations is that the five homologous proteins are single ion transporters. Presumably, MrpA transports Na+ whereas MrpD transports H+ in opposite directions, resulting in antiporter activity. This hypothesis has implications for the complex I functional mechanism, suggesting that one Na+ channel, NuoL, and two H+ channels, NuoM and NuoN, are present.

A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli--expression and characterization of cytochrome-tagged Complex I subunits.

Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane-spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C-terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c(550). Compared with other available fusion-protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter-like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo-cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c(550) domain in all the fusion proteins exhibited normal spectra and redox properties, with an E(m) of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c-tag. Finally, a his-tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins.

Photolabile ubiquinone analogues for identification and characterization of quinone binding sites in proteins.

Quinones are essential components in most cell and organelle bioenergetic processes both for direct electron and/or proton transfer reactions but also as means to regulate various bioenergetic processes by sensing cell redox states. To understand how quinones interact with proteins, it is important to have tools for identifying and characterizing quinone binding sites. In this work three different photo-reactive azidoquinones were synthesized, two of which are novel compounds, and the methods of synthesis was improved. The reactivity of the azidoquinones was first tested with model peptides, and the adducts formed were analyzed by mass spectrometry. The added mass detected was that of the respective azidoquinone minus N(2). Subsequently, the biological activity of the three azidoquinones was assessed, using three enzyme systems of different complexity, and the ability of the compounds to inactivate the enzymes upon illumination with long wavelength UV light was investigated. The soluble flavodoxin-like protein WrbA could only use two of the azidoquinones as substrates, whereas respiratory chain Complexes I and II could utilize all three compounds as electron acceptors. Complex II, purified in detergent, was very sensitive to illumination also in the absence of azidoquinones, making the 'therapeutic window' in that enzyme rather narrow. In membrane bound Complex I, only two of the compounds inactivated the enzyme, whereas illumination in the presence of the third compound left enzyme activity essentially unchanged. Since unspecific labeling should be equally effective for all the compounds, this demonstrates that the observed inactivation is indeed caused by specific labeling.

Electrical wiring of live, metabolically enhanced Bacillus subtilis cells with flexible osmium-redox polymers.

The present study explores genetic engineering of the respiratory chain and the application of two different flexible osmium redox polymers to achieve efficient electric communication between the gram-positive organism Bacillus subtilis and an electrode. Poly(1-vinylimidazole)(12)-[Os-(4,4'-dimethyl-2,2'-bipyridyl)(2)Cl(2)](+/2+) (osmium redox polymer I) and poly(vinylpyridine)-[Os-(N,N'-methylated-2,2'-biimidazole)(3)](2+/3+) (osmium redox polymer II) were investigated for efficient electrical "wiring" of viable gram-positive bacterial cells to electrodes. Using a B. subtilis strain that overproduces succinate/quinone oxidoreductase (respiratory complex II), we were able to improve the current response several fold using succinate as substrate, in both batch and flow analysis modes, and using gold and graphite electrodes. The efficiency of the osmium redox polymer, working as electron transfer mediator between the cells and the electrode, was compared with that of a soluble mediator (hexacyanoferrate). The results demonstrated that mediators did not have to pass the cytosolic membrane to bring about an efficient electronic communication between bacterial cells with a thick cell wall and electrodes.

Precise detection of pH inside large unilamellar vesicles using membrane-impermeable dendritic porphyrin-based nanoprobes.

Accurate real-time measurements of proton concentration gradients are pivotal to mechanistic studies of proton translocation by membrane-bound enzymes. Here we report a detailed characterization of the pH-sensitive fluorescent nanoprobe Glu(3), which is well suited for pH measurements in microcompartmentalized biological systems. The probe is a polyglutamic porphyrin dendrimer in which multiple carboxylate termini ensure its high water solubility and prevent its diffusion across phospholipid membranes. The probe's pK is in the physiological pH range, and its protonation can be followed ratiometrically by absorbance or fluorescence in the ultraviolet-visible spectral region. The usefulness of the probe was enhanced by using a semiautomatic titration system coupled to a charge-coupled device (CCD) spectrometer, enabling fast and accurate titrations and full spectral coverage of the system at millisecond time resolution. The probe's pK was measured in bulk solutions as well as inside large unilamellar vesicles in the presence of physiologically relevant ions. Glu(3) was found to be completely membrane impermeable, and its distinct spectroscopic features permit pH measurements inside closed membrane vesicles, enabling quantitative mechanistic studies of membrane-spanning proteins. Performance of the probe was demonstrated by monitoring the rate of proton leakage through the phospholipid bilayer in large vesicles with and without the uncoupler gramicidin present. Overall, as a probe for biological proton translocation measurements, Glu(3) was found to be superior to the commercially available pH indicators.

Direct and mediated electron transfer between intact succinate:quinone oxidoreductase from Bacillus subtilis and a surface modified gold electrode reveals redox state-dependent conformational changes.

Succinate:quinone oxidoreductase (SQR) from Bacillus subtilis consists of two hydrophilic protein subunits comprising succinate dehydrogenase, and a di-heme membrane anchor protein harboring two putative quinone binding sites, Q(p) and Q(d). In this work we have used spectroelectrochemistry to study the electronic communication between purified SQR and a surface modified gold capillary electrode. In the presence of two soluble quinone mediators the midpoint potentials of both hemes were revealed essentially as previously determined by conventional redox titration (heme b(H), E(m)=+65 mV, heme b(L), E(m)=-95 mV). In the absence of mediators the enzyme still communicated with the electrode, albeit with a reproducible hysteresis, resulting in the reduction of both hemes occurring approximately at the midpoint potential of heme b(L), and with a pronounced delay of reoxidation. When the specific inhibitor 2-n-heptyl-4 hydroxyquinoline N-oxide (HQNO), which binds to Q(d) in B. subtilis SQR, was added together with the two quinone mediators, rapid reductive titration was still possible which can be envisioned as an electron transfer occurring via the HQNO insensitive Q(p) site. In contrast, the subsequent oxidative titration was severely hampered in the presence of HQNO, in fact it completely resembled the unmediated reaction. If mediators communicate with Q(p) or Q(d), either event is followed by very rapid electron redistribution within the enzyme. Taken together, this strongly suggests that the accessibility of Q(p) depended on the redox state of the hemes. When both hemes were reduced, and Q(d) was blocked by HQNO, quinone-mediated communication via the Q(p) site was no longer possible, revealing a redox-dependent conformational change in the membrane anchor domain.

Transmembrane topology of FRO2, a ferric chelate reductase from Arabidopsis thaliana.

Iron uptake in Arabidopsis thaliana is mediated by ferric chelate reductase FRO2, a transmembrane protein belonging to the flavocytochrome b family. There is no high resolution structural information available for any member of this family. We have determined the transmembrane topology of FRO2 experimentally using the alkaline phosphatase fusion method. The resulting topology is different from that obtained by theoretical predictions and contains 8 transmembrane helices, 4 of which build up the highly conserved core of the protein. This core is present in the entire flavocytochrome b family. The large water soluble domain of FRO2, which contains NADPH, FAD and oxidoreductase sequence motifs, was located on the inside of the membrane.

The mitochondrial and prokaryotic proton-translocating NADH:ubiquinone oxidoreductases: similarities and dissimilarities of the quinone-junction sites.

The catalytic properties of the rotenone-sensitive NADH:ubiquinone reductase (Complex I) in bovine heart submitochondrial particles and in inside-out vesicles derived from Paracoccus denitrificans and Rhodobacter capsulatus were compared. The prokaryotic enzymes catalyze the NADH oxidase and NADH:quinone reductase reactions with similar kinetic parameters as those for the mammalian Complex I, except for lower apparent affinities for the substrates--nucleotides. Unidirectional competitive inhibition of NADH oxidation by ADP-ribose, previously discovered for submitochondrial particles, was also evident for tightly coupled P. denitrificans vesicles, thus suggesting that a second, NAD(+)-specific site is present in the simpler prokaryotic enzyme. The inhibitor sensitivity of the forward and reverse electron transfer reactions was compared. In P. denitrificans and Bos taurus vesicles different sensitivities to rotenone and Triton X-100 for the forward and reverse electron transfer reactions were found. In bovine heart preparations, both reactions showed the same sensitivity to piericidin, and the inhibition was titrated as a straight line. In P. denitrificans, the forward and reverse reactions show different sensitivity to piericidin and the titrations of both activities were curvilinear with apparent I(50) (expressed as mole of inhibitor per mole of enzyme) independent of the enzyme concentration. This behavior is explained by a model involving two different sites rapidly interacting with piericidin within the hydrophobic phase.

The 'antiporter module' of respiratory chain complex I includes the MrpC/NuoK subunit -- a revision of the modular evolution scheme.

Respiratory chain Complex I or NADH:quinone oxidoreductase catalyzes oxidation of NADH in the mitochondrial matrix or bacterial cytoplasm and reduction of quinone in the membrane, coupled to pumping of 4H(+)/2e(-) across the membrane. The same enzyme complex is also capable of the reverse reaction, i.e. Deltamu(H(+))-supported NAD(+) reduction. The molecular mechanism that couples electron transfer to proton pumping is not understood. The Complex I enzyme, containing 14 protein subunits necessary for function, has evolved from smaller functional building blocks. Three Complex I protein subunits, NuoL, NuoM and NuoN, show primary sequence similarity to one particular class of antiporters, and are thus predicted to play a role in the proton translocation machinery. These antiporters, MrpA and MrpD are encoded by a conserved gene cluster, that contains seven genes. In previous work we have determined that these antiporters come in two subclasses, MrpA-type and MrpD-type, and that the Complex I subunit NuoL is more closely related to MrpA and NuoM and N are more closely related to the MrpD antiporter. This implied that both MrpA and MrpD had been recruited to Complex I, rather than arising from gene duplications of one antiporter encoding gene. In this work we show that MrpC and NuoK are homologous proteins. The most plausible explanation for these findings is that a multisubunit antiporter complex was recruited to the ancestral enzyme. We further conclude that the last common ancestor of the Complex I enzyme family and membrane bound NiFe hydrogenases of type 3 and 4 contained the NuoKLMN subunit module.