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ABSTRACT: Shewanella spp. demonstrate great variability in the use of terminal electron acceptors in anaerobic respiration; these include nitrate, fumarate, DMSO, trimethylamine oxide, sulphur compounds and metal oxides. These pathways open up possible applications in bioremediation. The wide variety of respiratory substrates for Shewanella is correlated with the evolution of several multi-haem membrane-bound, periplasmic and outer-membrane c-type cytochromes. The 21 kDa c-type cytochrome CymA of the freshwater strain Shewanella oneidensis MR-1 has an N-terminal membrane anchor and a globular tetrahaem periplasmic domain. According to sequence alignments, CymA is a member of the NapC/NirT family. This family of redox proteins is responsible for electron transfer from the quinone pool to periplasmic and outer-membrane-bound reductases. Prior investigations have shown that the absence of CymA results in loss of the ability to respire with Fe(III), fumarate and nitrate, indicating that CymA is involved in electron transfer to several terminal reductases. Here we describe the expression, purification and characterization of a soluble, truncated CymA ('CymA). Potentiometric studies suggest that there are two pairs of haems with potentials of -175 and -261 mV and that 'CymA is an efficient electron donor for the soluble fumarate reductase, flavocytochrome c(3).
Biochemical Society Transactions 09/2002; 30(4):658-62. · 3.71 Impact Factor
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ABSTRACT: Flavocytochrome b(2) catalyzes the oxidation of L-lactate to pyruvate and the transfer of electrons to cytochrome c. The enzyme consists of a flavin-binding domain, which includes the active site for lacate oxidation, and a b(2)-cytochrome domain, required for efficient cytochrome c reduction. To better understand the structure and function of intra- and interprotein electron transfer, we have determined the crystal structure of the independently expressed flavin-binding domain of flavocytochrome b(2) to 2.50 A resolution and compared this with the structure of the intact enzyme, redetermined at 2.30 A resolution, both structures being from crystals cooled to 100 K. Whereas there is little overall difference between these structures, we do observe significant local changes near the interface region, some of which impact on amino acid side chains, such as Arg289, that have been shown previously to have an important role in catalysis. The disordered loop region found in flavocytochrome b(2) and its close homologues remain unresolved in frozen crystals of the flavin-binding domain, implying that the presence of the b(2)-cytochrome domain is not responsible for this positional disorder. The flavin-binding domain interacts poorly with cytochrome c, but we have introduced acidic residues in the interdomain interface region with the aim of enhancing cytochrome c binding. While the mutations L199E and K201E within the flavin-binding domain resulted in unimpaired lactate dehydrogenase activity, they failed to enhance electron-transfer rates with cytochrome c. This is most likely due to the disordered loop region obscuring all or part of the surface having the potential for productive interaction with cytochrome c.
Biochemistry 05/2002; 41(13):4264-72. · 3.42 Impact Factor
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ABSTRACT: Site-directed mutants of the phylogenetically conserved phenylalanine residue F393 were constructed in flavocytochrome P450 BM3 from Bacillus megaterium. The high degree of conservation of this residue in the P450 superfamily and its proximity to the heme (and its ligand Cys400) infers an essential role in P450 activity. Extensive kinetic and thermodynamic characterization of mutant enzymes F393A, F393H, and F393Y highlighted significant differences from wild-type P450 BM3. All enzymes expressed to high levels and contained their full complement of heme. While the reduction and subsequent treatment of the mutant P450s with carbon monoxide led to the formation of the characteristic P450 spectra in all cases, the absolute position of the Soret absorption varied across the series WT/F393Y (449 nm), F393H (445 nm), and F393A (444 nm). Steady-state turnover rates with both laurate and arachidonate showed the trend WT > F393Y > F393H > F393A. Conversely, the trend in the pre-steady-state flavin-to-heme electron transfer was the reverse of the steady-state scenario, with rates varying F393A > F393H > F393Y approximately wild-type. These data are consistent with the more positive substrate-free [-312 mV (F393A), -332 mV (F393H)] and substrate-bound [-151 mV (F393A), -176 mV (F393H)] reduction potentials of F393A and F393H heme domains, favoring the stabilization of the ferrous-form in the mutant P450s relative to wild-type. Elevation of the heme iron reduction potential in the F393A and F393H mutants facilitates faster electron transfer to the heme. This results in a decrease in the driving force for oxygen reduction by the ferrous heme iron, so explaining lower overall turnover of the mutant P450s. We postulate that the nature of the residue at position 393 is important in controlling the delicate equilibrium observed in P450s, whereby a tradeoff is established between the rate of heme reduction and the rate at which the ferrous heme can bind and, subsequently, reduce molecular oxygen.
Biochemistry 11/2001; 40(45):13421-9. · 3.42 Impact Factor
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ABSTRACT: There is now overwhelming evidence supporting a common mechanism for fumarate reduction in the respiratory fumarate reductases. The X-ray structures of substrate-bound forms of these enzymes indicate that the substrate is well positioned to accept a hydride from FAD and a proton from an arginine side chain. Recent work on the enzyme from Shewanella frigidimarina [Doherty, M. K., Pealing, S. L., Miles, C. S., Moysey, R., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2000) Biochemistry 39, 10695-10701] has strengthened the assignment of an arginine (Arg402) as the proton donor in fumarate reduction. Here we describe the crystallographic and kinetic analyses of the R402A, R402K, and R402Y mutant forms of the Shewanella enzyme. The crystal structure of the R402A mutant (2.0 A resolution) shows it to be virtually identical to the wild-type enzyme, apart from the fact that a water molecule occupies the position previously taken by part of the guanidine group of R402. Although structurally similar to the wild-type enzyme, the R402A mutant is inactive under all the conditions that were studied. This implies that a water molecule, in this position in the active site, cannot function as the proton donor for fumarate reduction. In contrast to the R402A mutation, both the R402K and R402Y mutant enzymes are active. Although this activity was at a very low level (at pH 7.2 some 10(4)-fold lower than that for the wild type), it does imply that both lysine and tyrosine can fulfill the role of an active site proton donor, albeit very poorly. The crystal structures of the R402K and R402Y mutant enzymes (2.0 A resolution) show that distances from the lysine and tyrosine side chains to the nearest carbon atom of fumarate are approximately 3.5 A, clearly permitting proton transfer. The combined results from mutagenesis, crystallographic, and kinetic studies provide formidable evidence that R402 acts as both a Lewis acid (stabilizing the build-up of negative charge upon hydride transfer from FAD to fumarate) and a Brønsted acid (donating the proton to the substrate to complete the formation of succinate).
Biochemistry 11/2001; 40(41):12292-8. · 3.42 Impact Factor
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ABSTRACT: The bioI gene has been sub-cloned and over-expressed in Escherichia coli, and the protein purified to homogeneity. The protein is a cytochrome P450, as indicated by its visible spectrum (low-spin haem iron Soret band at 419 nm) and by the characteristic carbon monoxide-induced shift of the Soret band to 448 nm in the reduced form. N-terminal amino acid sequencing and mass spectrometry indicate that the initiator methionine is removed from cytochrome P450 BioI and that the relative molecular mass is 44,732 Da, consistent with that deduced from the gene sequence. SDS-PAGE indicates that the protein is homogeneous after column chromatography on DE-52 and hydroxyapatite, followed by FPLC on a quaternary ammonium ion-exchange column (Q-Sepharose). The purified protein is of mixed spin-state by both electronic spectroscopy and by electron paramagnetic resonance [g values=2.41, 2.24 and 1.97/1.91 (low-spin) and 8.13, 5.92 and 3.47 (high-spin)]. Magnetic circular dichroism and electron paramagnetic resonance studies indicate that P450 BioI has a cysteine-ligated b-type haem iron and the near-IR magnetic circular dichroism band suggests strongly that the sixth ligand bound to the haem iron is water. Resonance Raman spectroscopy identifies vibrational signals typical of cytochrome P450, notably the oxidation state marker v4 at 1,373 cm(-1) (indicating ferric P450 haem) and the splitting of the spin-state marker v3 into two components (1,503 cm(-1) and 1,488 cm(-1)), indicating cytochrome P450 BioI to be a mixture of high- and low-spin forms. Fatty acids were found to bind to cytochrome P450 BioI, with myristic acid (Kd=4.18+/-0.26 microM) and pentadecanoic acid (Kd=3.58+/-0.54 microM) having highest affinity. The fatty acid analogue inhibitor 12-imidazolyldodecanoic acid bound extremely tightly (Kd<1 microM), again indicating strong affinity for fatty acid chains in the P450 active site. Catalytic activity was demonstrated by reconstituting the P450 with either a soluble form of human cytochrome P450 reductase, or a Bacillus subtilis ferredoxin and E. coli ferredoxin reductase. Substrate hydroxylation at the omega-terminal position was demonstrated by turnover of the chromophoric fatty acid para-nitrophenoxydodecanoic acid, and by separation of product from the reaction of P450 BioI with myristic acid.
JBIC Journal of Biological Inorganic Chemistry 07/2001; 6(5-6):523-33. · 3.29 Impact Factor
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ABSTRACT: The tetrahaem cytochrome isolated during anaerobic growth of Shewanella frigidimarina NCIMB400 is a small protein (86 residues) involved in electron transfer to Fe(III), which can be used as a terminal respiratory oxidant by this bacterium. A 3D solution structure model of the reduced form of the cytochrome has been determined using NMR data in order to determine the relative orientation of the haems. The haem core architecture of S. frigidimarina tetrahaem cytochrome differs from that found in all small tetrahaem cytochromes c(3) so far isolated from strict anaerobes, but has some similarity to the N-terminal cytochrome domain of flavocytochrome c(3) isolated from the same bacterium. NMR signals obtained for the four haems of S. frigidimarina tetrahaem cytochrome at all stages of oxidation were cross-assigned to the solution structure using the complete network of chemical exchange connectivities. Thus, the order in which each haem in the structure becomes oxidised was determined.
FEBS Letters 02/2001; 489(1):8-13. · 3.54 Impact Factor
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ABSTRACT: Bacillus megaterium P450 BM3 is a fatty acid hydroxylase with selectivity for long chain substrates (C(12)-C(20)). Binding or activity with substrates of chain length <C(12) has not been reported. Rational mutagenesis was used to re-design the enzyme to encourage binding of short chain fatty acids (C(4)-C(10)). We show that wild-type P450 BM3 has activity and weak affinity for substrates as short as butyrate (C(4)). However, turnover/binding of short chain substrates is dramatically increased by introducing a novel substrate carboxylate binding site close to the heme. Mutant L181K shows catalytic efficiency (k(cat)/K(M)) increased >13-fold with butyrate, while the L75T/L181K double mutant has k(cat)/K(M) increased >15-fold with hexanoate and binding (K(d)) improved >28-fold for butyrate. Removing the arginine 47/lysine 51 carboxylate binding motif at the mouth of the active site disfavours binding of all fatty acids, indicating its importance in the initial recognition of substrates.
FEBS Letters 12/2000; 486(2):173-7. · 3.54 Impact Factor
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ABSTRACT: Substitution by cysteine of one of the heme iron axial ligands (His66) of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase from Saccharomyces cerevisiae) has resulted in an enzyme (H66C-b2) which remains a competent L-lactate dehydrogenase (kcat 272+/-6 s(-1), L-lactate KM 0.60+/-0.06 mM, 25 degrees C, I 0.10, Tris-HCl, pH 7.5) but which has no cytochrome c reductase activity. As a result of the mutation, the reduction potential of the heme was found to be -265+5 mV, over 240 mV more negative than that of the wild-type enzyme, and therefore unable to be reduced by L-lactate. Surface-enhanced resonance Raman spectroscopy indicates similarities between the heme of H66C-b2 and those of cytochromes P450, with a nu4 band at 1,345 cm(-1) which is indicative of cysteine heme-iron ligation. In addition, EPR spectroscopy yields g-values at 2.33, 2.22 and 1.94, typical of low-spin ferric cytochromes P450, optical spectra show features between 600 and 900 nm which are characteristic of sulfur coordination of the heme iron, and MCD spectroscopy shows a blue-shifted NIR CT band relative to the wild-type, implying that the H66C-b2 heme is P450-like. Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.
JBIC Journal of Biological Inorganic Chemistry 11/2000; 5(5):584-92. · 3.29 Impact Factor
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ABSTRACT: The active sites of respiratory fumarate reductases are highly conserved, indicating a common mechanism of action involving hydride and proton transfer. Evidence from the X-ray structures of substrate-bound fumarate reductases, including that for the enzyme from Shewanella frigidimarina [Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112], indicates that the substrate is well positioned to accept a hydride from N5 of the FAD. However, the identity of the proton donor has been the subject of recent debate and has been variously proposed to be (using numbering for the S. frigidimarina enzyme) His365, His504, and Arg402. We have used site-directed mutagenesis to examine the roles of these residues in the S. frigidimarina enzyme. The H365A and H504A mutant enzymes exhibited lower k(cat) values than the wild-type enzyme but only by factors of 3-15, depending on pH. This, coupled with the increase in K(m) observed for these enzymes, indicates that His365 and His504 are involved in Michaelis complex formation and are not essential catalytic residues. In fact, examination of the crystal structure of S. frigidimarina fumarate reductase has led to the proposal that Arg402 is the only plausible active site acid. Consistent with this proposal, we report that the R402A mutant enzyme has no detectable fumarate reductase activity. The crystal structure of the H365A mutant enzyme shows that, in addition to the replacement at position 365, there have been some adjustments in the positions of active site residues. In particular, the observed change in the orientation of the Arg402 side chain could account for the decrease in k(cat) seen with the H365A enzyme. These results demonstrate that an active site arginine and not a histidine residue is the proton donor for fumarate reduction.
Biochemistry 10/2000; 39(35):10695-701. · 3.42 Impact Factor
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ABSTRACT: In the absence of oxygen many bacteria are able to utilise fumarate as a terminal oxidant for respiration. In most known organisms the fumarate reductases are membrane-bound iron-sulfur flavoproteins but Shewanella species produce a soluble, periplasmic flavocytochrome c(3) that catalyses this reaction. The active sites of all fumarate reductases are clearly conserved at the structural level, indicating a common mechanism. The structures of fumarate reductases from two Shewanella species have been determined. Fumarate, succinate and a partially hydrated fumarate ligand are found in equivalent locations in different crystals, tightly bound in the active site and close to N5 of the FAD cofactor, allowing identification of amino acid residues that are involved in substrate binding and catalysis. Conversion of fumarate to succinate requires hydride transfer from FAD and protonation by an active site acid. The identity of the proton donor has been open to question but we have used structural considerations to suggest that this function is provided by an arginine side chain. We have confirmed this experimentally by analysing the effects of site-directed mutations on enzyme activity. Substitutions of Arg402 lead to a dramatic loss of activity whereas neither of the two active site histidine residues is required for catalysis.
Biochimica et Biophysica Acta 09/2000; 1459(2-3):310-5. · 4.66 Impact Factor
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ABSTRACT: Shewanella frigidimarina NCIMB400 is a non-fermenting, facultative anaerobe from the gamma group of proteobacteria. When grown anaerobically this organism produces a wide variety of periplasmic c-type cytochromes, mostly of unknown function. We have purified a small, acidic, low-potential tetrahaem cytochrome with similarities to the cytochromes c(3) from sulphate-reducing bacteria. The N-terminal sequence was used to design PCR primers and the cctA gene encoding cytochrome c(3) was isolated and sequenced. The EPR spectrum of purified cytochrome c(3) indicates that all four haem irons are ligated by two histidine residues, a conclusion supported by the presence of eight histidine residues in the polypeptide sequence, each of which is conserved in a related cytochrome c(3) and in the cytochrome domains of flavocytochromes c(3). All four haems exhibit low midpoint redox potentials that range from -207 to -58 mV at pH 7; these values are not significantly influenced by pH changes. Shewanella cytochrome c(3) consists of a mere 86 amino acid residues with a predicted molecular mass of 11780 Da, including the four attached haem groups. This corresponds closely to the value of 11778 Da estimated by electrospray MS. To examine the function of this novel cytochrome c(3) we constructed a null mutant by gene disruption. S. frigidimarina lacking cytochrome c(3) grows well aerobically and its growth rate under anaerobiosis with a variety of electron acceptors is indistinguishable from that of the wild-type parent strain, except that respiration with Fe(III) as sole acceptor is severely, although not completely, impaired.
Biochemical Journal 08/2000; 349(Pt 1):153-8. · 4.90 Impact Factor
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ABSTRACT: Flavocytochrome b(2) from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction. The crystal structure of the native yeast enzyme has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863] as well as that of the sulfite adduct of the recombinant enzyme produced in Escherichia coli [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303-313]; several key active site residues were identified. In the sulfite adduct crystal structure, Arg289 adopts two alternative conformations. In one of them, its side chain is stacked against that of Arg376, which interacts with the substrate; in the second orientation, the R289 side chain points toward the active site. This residue has now been mutated to lysine and the mutant enzyme, R289K-b(2), characterized kinetically. Under steady-state conditions, kinetic parameters (including the deuterium kinetic isotope effect) indicate the mutation affects k(cat) by a factor of about 10 and k(cat)/K(M) by up to nearly 10(2). Pre-steady-state kinetic analysis of flavin and heme reduction by lactate demonstrates that the latter is entirely limited by flavin reduction. Inhibition studies on R289K-b(2) with a range of compounds show a general rise in K(i) values relative to that of wild-type enzyme, in line with the elevation of the K(M) for L-lactate in R289K-b(2); they also show a change in the pattern of inhibition by pyruvate and oxalate, as well as a loss of the inhibition by excess substrate. Altogether, the kinetic studies indicate that the mutation has altered the first step of the catalytic cycle, namely, flavin reduction; they suggest that R289 plays a role both in Michaelis complex and transition-state stabilization, as well as in ligand binding to the active site when the flavin is in the semiquinone state. In addition, it appears that the mutation has not affected electron transfer from fully reduced flavin to heme, but may have slowed the second intramolecular ET step, namely, transfer from flavin semiquinone to heme b(2). Finally, the X-ray crystal structure of R289K-b(2), with sulfite bound at the active site, has been determined to 2.75 A resolution. The lysine side chain at position 289 is well-defined and in an orientation that corresponds approximately to one of the alternative conformations observed in the structure of the recombinant enzyme-sulfite complex [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303-313]. Comparisons between the R289K-b(2) and wild-type structures allow the kinetic results to be interpreted in a structural context.
Biochemistry 04/2000; 39(12):3266-75. · 3.42 Impact Factor
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ABSTRACT: The 1.8 A resolution crystal structure of the tetraheme flavocytochrome c3, Fcc3, provides the first mechanistic insight into respiratory fumarate reductases or succinate dehydrogenases. The multi-redox center, three-domain protein shows a 40 A long 'molecular wire' allowing rapid conduction of electrons through a new type of cytochrome domain onto the active site flavin, driving the reduction of fumarate to succinate. In this structure a malate-like molecule is trapped in the enzyme active site. The interactions between this molecule and the enzyme suggest a clear mechanism for fumarate reduction in which the substrate is polarized and twisted, facilitating hydride transfer from the reduced flavin and subsequent proton transfer. The enzyme active site in the oxidized form is completely buried at the interface between the flavin-binding and the clamp domains. Movement of the cytochrome and clamp domains is postulated to allow release of the product.
Natural Structural Biology 01/2000; 6(12):1108-12.
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ABSTRACT: The fumarate reductase (flavocytochrome c(3)) from Shewanella frigidimarina (formerly S. putrefaciens) NCIMB400 has been crystallized in the space group P2(1), with cell dimensions of a = 45.447 A, b = 92.107 A, c = 78.311 A, and beta = 91.038 degrees and one molecule per asymmetric unit. A native data set has been collected to 1.8 A. The gene encoding Fcc(3) from the S. frigidimarina type strain ACAM591 has been cloned and sequenced and the protein crystallized in space group P2(1) with cell dimensions of a = 45.359 A, b = 88.051 A, c = 77.473 A, and beta = 104.499 degrees. Anomalous data have also been collected from the NCIMB400 crystal allowing the heme iron positions to be identified.
Journal of Structural Biology 09/1999; 127(1):76-8. · 3.41 Impact Factor
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ABSTRACT: The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 microM, kcat 3960 min-1; Y51F mutant, Km 432 microM, kcat 6140 min-1; wild-type, Km 288 microM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (DeltaG) resulting from a smaller DeltaG of substrate binding. The side chain of Phe-42 acts as a phenyl 'cap' over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2. 08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 microM, kcat 14620 min-1; compared with values of 4.7 microM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a 'fast' to a 'slow' rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat 'fast', 760 (1620) min-1; kcat 'slow', 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole. Mutant F87G binds 1-phenylimidazole >10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid >30-fold more tightly than wild-type.
Biochemical Journal 04/1999; 339 ( Pt 2):371-9. · 4.90 Impact Factor
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ABSTRACT: The thermodynamic and catalytic properties of flavocytochrome c3 from Shewanella frigidimarina have been studied using a combination of protein film voltammetry and solution methods. As measured by solution kinetics, maximum catalytic efficiencies for fumarate reduction (kcat/Km = 2.1 x 10(7) M-1 s-1 at pH 7.2) and succinate oxidation (kcat/Km = 933 M-1 s-1 at pH 8.5) confirm that flavocytochrome c3 is a unidirectional fumarate reductase. Very similar catalytic properties are observed for the enzyme adsorbed to monolayer coverage at a pyrolytic graphite "edge" electrode, thus confirming the validity of the electrochemical method for providing complementary information. In the absence of fumarate, the adsorbed enzyme displays a complex envelope of reversible redox signals which can be deconvoluted to yield the contributions from each active site. Importantly, the envelope is dominated by the two-electron signal due to FAD [E degrees ' = -152 mV vs the standard hydrogen electrode (SHE) at pH 7.0 and 24 degrees C] which enables quantitative examination of this center, the visible spectrum of which is otherwise masked by the intense absorption bands due to the hemes. The FAD behaves as a cooperative two-electron center with a pH-dependent reduction potential that is modulated (pKox at 6.5) by ionization of a nearby residue. In conjunction with the kinetic pKa values determined for the forward and reverse reactions (7.4 and 8.6, respectively), a mechanism for fumarate reduction, incorporating His365 and an anionic form of reduced FAD, is proposed. The reduction potentials of the four heme groups, estimated by analysis of the underlying envelope, are -102, -146, -196, and -238 mV versus the SHE at pH 7.0 and 24 degrees C and are comparable to those determined by redox potentiometry.
Biochemistry 04/1999; 38(11):3302-9. · 3.42 Impact Factor
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Biochemical Society Transactions 03/1999; 27(2):185-9. · 3.71 Impact Factor
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Biochemical Society Transactions 03/1999; 27(2):190-6. · 3.71 Impact Factor
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ABSTRACT: Dissimilatory Fe(III) reduction by Shewanella putrefaciens and related species has generated considerable interest in biochemical characterization of the pathways for anaerobic electron transfer in this organism. Two strains, MR-1 and NCIMB 400, have been extensively used, and several respiratory enzymes have been isolated from each. It has become apparent that significant sequence differences exist between homologous proteins from these strains. The 16S rRNA from NCIMB 400 was sequenced and compared to the sequences from MR-1 and other Shewanella strains. The results indicate that NCIMB 400 is significantly more closely related to the newly identified Shewanella frigidimarina than to the S. putrefaciens type strain. It is therefore proposed that NCIMB 400 should be reclassified as S. frigidimarina.
International journal of systematic bacteriology 02/1999; 49 Pt 1:189-91.
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Biochemical Society Transactions 09/1998; 26(3):418-21. · 3.71 Impact Factor
