Bacterial Phosphating of Mild (Unalloyed) Steel

Mild (unalloyed) steel electrodes were incubated in phosphate-buffered cultures of aerobic, biofilm-forming Rhodococcus sp. strain C125 and Pseudomonas putida mt2. A resulting surface reaction leading to the formation of a corrosion-inhibiting vivianite layer was accompanied by a characteristic electrochemical potential (E) curve. First, E increased slightly due to the interaction of phosphate with the iron oxides covering the steel surface. Subsequently, E decreased rapidly and after 1 day reached −510 mV, the potential of free iron, indicating the removal of the iron oxides. At this point, only scattered patches of bacteria covered the surface. A surface reaction, in which iron was released and vivianite precipitated, started. E remained at −510 mV for about 2 days, during which the vivianite layer grew steadily. Thereafter, E increased markedly to the initial value, and the release of iron stopped. Changes in E and formation of vivianite were results of bacterial activity, with oxygen consumption by the biofilm being the driving force. These findings indicate that biofilms may protect steel surfaces and might be used as an alternative method to combat corrosion.     

Sedimentary Sulphur:Iron Ratio Indicates Vivianite Occurrence: A Study from Two Contrasting Freshwater Systems

An increasing number of studies constrain the importance of iron for the long-term retention of phosphorus (P) under anoxic conditions, i.e. the formation of reduced iron phosphate minerals such as vivianite (Fe₃(PO₄)₂⋅8H₂O). Much remains unknown about vivianite formation, the factors controlling its occurrence, and its relevance for P burial during early sediment diagenesis. To study the occurrence of vivianite and to assess its relevance for P binding, surface sediments of two hydrologically contrasting waters were analysed by heavy-liquid separation and subsequent powder X-ray diffraction. In Lake Arendsee, vivianite was present in deeper sediment horizons and not in the uppermost layers with a sharp transition between vivianite and non-vivianite bearing layers. In contrast, in lowland river Lower Havel vivianite was present in the upper sediment layers and not in deeper horizons with a gradual transition between non-vivianite and vivianite bearing layers. In both waters, vivianite occurrence was accompanied by the presence of pyrite (FeS₂). Vivianite formation was favoured by an elevated iron availability through a lower degree of sulphidisation and was present at a molar ratio of total sulphur to reactive iron smaller than 1.1, only. A longer lasting burden of sediments by organic matter, i.e. due to eutrophication, favours the release of sulphides, and the formation of insoluble iron sulphides leading to a lack of available iron and to less or no vivianite formation. This weakening in sedimentary P retention, representing a negative feedback mechanism (P release) in terms of water quality, could be partly compensated by harmless Fe amendments.     

2,4-二氯苯酚的矿物表面化学行为研究

矿物质是土壤中的重要组成成分,本实验采用动态反应和平衡反应研究氧化物矿物表面2,4-二氯苯酚(2,4-DCP)的化学行为.结果表明,无定形铁氧化物、针铁矿、α-MnO₂、δ-MnO₂对自然的或外源的有机化合物有较强的氧化能力,其中对2,4二氯苯酚的氧化能力是:δ-MnO₂αMnO₂>无定形铁氧化物、针铁矿.采用分段法估算反应系统的速率,发现氧化锰氧化2,4-二氯苯酚的反应速率是氧化铁的3～50倍,δ-MnO₂氧化2,4-二氯苯酚的速率是α-MnO₂的1.5～3.2倍.根系分泌物由于参与氧化物表面化学反应,对系统2,4-二氯苯酚的氧化过程产生一定影响,α-MnO₂加入根系分泌物后,2,4-二氯苯酚在溶液中的消减速率明显减慢,δ-MnO₂则相反,在根系分泌物共存下溶液中的2,4-二氯苯酚的转化率增加.Cu复合共存可能影响2,4二氯苯酚在矿物表面的键合氧化过程,其中δ-MnO₂对铜吸附能力大于αMnO₂,Cu复合共存对δMnO₂表面2,4-二氯苯酚的氧化影响较大.     

The effect of inorganic phosphate and hydrogenase on the corrosion of mild steel

The data reported in this investigation revealed that phosphate and hydrogenase can affect the corrosion of mild steel. Phosphate reacted on mild-steel with concomitant evolution of hydrogen gas (H₂) and the formation of vivianite. The enzyme hydrogenase was shown to accelerate this reaction by oxidizing the H₂ produced, its known substrate. The components of biological growth media, one of which has been identified as phosphate, can interact with mild steel and cause corrosion. The implications of these findings to the corrosion industry are discussed. Correspondence to: E. Laishley     

Characterization of a new dihemic c(4)-type cytochrome isolated from Thiobacillus ferrooxidans

A new soluble c-type cytochrome has been purified to homogeneity from the acidophilic proteobacterium Thiobacillus ferrooxidans BRGM. It is characterized by an alpha-peak wavelength of 552 nm, a molecular mass of 26 567 Da (as determined by mass spectroscopy) and a pI value of 8. Optical redox titrations at pH 4.0 revealed the presence of two distinguishable redox species with an E(m) of 510 mV and an E(m) of 430 +/- 20 mV. EPR spectra recorded for this heme protein demonstrated the presence of stoichiometric amounts of two low-spin hemes with a g(z)() of 3.08 (510 mV species) and a g(z)() of 3.22 (430 mV species). Modifications of the physicochemical properties of the cytochrome were observed on complex formation with the blue copper protein rusticyanin, another soluble electron carrier in the genus Thiobacillus. N-Terminal sequencing yielded the polypeptide sequence up to the 50th residue. The determined sequence was found to be present (at 100% amino acid identity) in the (unfinished) genome of T. ferrooxidans ATCC 23270, and the corresponding full-length protein turned out to be surprisingly similar (34.5% amino acid identity) to another c(4)-type diheme protein from T. ferrooxidans BRGM [Cavazza, C., et al. (1996) Eur. J. Biochem. 242, 308-314], the gene of which is also present (at 97% amino acid identity) in the T. ferrooxidans ATCC 23270 genome. The physicochemical properties and sequence characteristics of both c(4) cytochromes present in the same bacteria are compared, and the functional role of this new diheme protein in the iron(II)-oxidizing electron transport chain in the genus Thiobacillus is discussed.     

鱼肝铁蛋白铁核表层接受电子能力的研究

采用直接电化学技术研究Hong鱼肝铁蛋白（Liver Ferritin of Dasyatis akajei,DALF）铁核表层接受还原电子的快慢速率和释放铁的动力学级数及规律。实验结果表明,在有氧环境下,DALF铁核表层以两相行为的方式快速地从铂金电极上获得还原电子且用于释放铁反应,其释放铁的还原电们分别为－125mV－375mV(vs.NHE,下同）。在控制还原电位为－200mV和－500mV的条件下,DALF铁核表层解放铁的速率分别为11．1Fe^3 /(DALF.min)和33．3Fe^3 (DALF.min）,因而认为DALF从铂金电极接受电子和解放铁的速率快慢与还原电位高低有关。血红素不仅能络合于DALF蛋白壳（DALFh）上,而且还能加速DALFh释放铁的速率,但无法增加DALFh释放铁的总量。DALF铁核结构中的磷铁组成存在着非均匀性。DALF铁核表层磷铁结构具有接受来自于蛋白壳电子隧道提供的还原电子能力。     

The effect of substrate, dihydrobiopterin, and dopamine on the EPR spectroscopic properties and the midpoint potential of the catalytic iron in recombinant human phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH(4)) and non-heme iron-dependent enzyme that hydroxylates L-Phe to L-Tyr. The paramagnetic ferric iron at the active site of recombinant human PAH (hPAH) and its midpoint potential at pH 7.25 (E(m)(Fe(III)/Fe(II))) were studied by EPR spectroscopy. Similar EPR spectra were obtained for the tetrameric wild-type (wt-hPAH) and the dimeric truncated hPAH(Gly(103)-Gln(428)) corresponding to the "catalytic domain." A rhombic high spin Fe(III) signal with a g value of 4.3 dominates the EPR spectra at 3.6 K of both enzyme forms. An E(m) = +207 +/- 10 mV was measured for the iron in wt-hPAH, which seems to be adequate for a thermodynamically feasible electron transfer from BH(4) (E(m) (quinonoid-BH(2)/BH(4)) = +174 mV). The broad EPR features from g = 9.7-4.3 in the spectra of the ligand-free enzyme decreased in intensity upon the addition of L-Phe, whereas more axial type signals were observed upon binding of 7,8-dihydrobiopterin (BH(2)), the stable oxidized form of BH(4), and of dopamine. All three ligands induced a decrease in the E(m) value of the iron to +123 +/- 4 mV (L-Phe), +110 +/- 20 mV (BH(2)), and -8 +/- 9 mV (dopamine). On the basis of these data we have calculated that the binding affinities of L-Phe, BH(2), and dopamine decrease by 28-, 47-, and 5040-fold, respectively, for the reduced ferrous form of the enzyme, with respect to the ferric form. Interestingly, an E(m) value comparable with that of the ligand-free, resting form of wt-hPAH, i.e. +191 +/- 11 mV, was measured upon the simultaneous binding of both L-Phe and BH(2), representing an inactive model for the iron environment under turnover conditions. Our findings provide new information on the redox properties of the active site iron relevant for the understanding of the reductive activation of the enzyme and the catalytic mechanism.     

Coupled Dynamics of Iron and Phosphorus in Sediments of an Oligotrophic Coastal Basin and the Impact of Anaerobic Oxidation of Methane

Studies of phosphorus (P) dynamics in surface sediments of lakes and coastal seas typically emphasize the role of coupled iron (Fe), sulfur (S) and P cycling for sediment P burial and release. Here, we show that anaerobic oxidation of methane (AOM) also may impact sediment P cycling in such systems. Using porewater and sediment profiles for sites in an oligotrophic coastal basin (Bothnian Sea), we provide evidence for the formation of Fe-bound P (possibly vivianite; Fe₃(PO₄)₂ ^.8H₂O) below the zone of AOM with sulfate. Here, dissolved Fe²⁺ released from oxides is no longer scavenged by sulfide and high concentrations of both dissolved Fe²⁺ (>1 mM) and PO₄ in the porewater allow supersaturation with respect to vivianite to be reached. Besides formation of Fe(II)-P, preservation of Fe-oxide bound P likely also contributes to permanent burial of P in Bothnian Sea sediments. Preliminary budget calculations suggest that the burial of Fe-bound P allows these sediments to act as a major sink for P from the adjacent eutrophic Baltic Proper.     

Distribution of Purple Photosynthetic Bacteria in Wetland and Woodland Habitats of Central and Northern Minnesota

Enrichment cultures for purple nonsulfur and sulfur photosynthetic bacteria were prepared from soil samples collected in central and northern Minnesota. The purple nonsulfur bacteria were found in most wetland soils sampled but were uncommon in woodland and grassland soils. The pH range of the soils in which these bacteria occurred was 3.8 to 7.8, and the oxidation-reduction potential (E(h)) range was +510 to -65 mV. Soils with a pH below 5.0 or an E(h) above +370 mV had few purple nonsulfur bacteria (<10/g of soil). Rhodopseudomonas viridis, a photosynthetic bacterium containing bacteriochlorophyll b, and the purple sulfur bacteria were common only in low-acidity wetland soils that were usually being reduced.     

Heme redox potential control in de novo designed four-alpha-helix bundle proteins

The effects of various mechanisms of metalloporphyrin reduction potential modulation were investigated experimentally using a robust, well-characterized heme protein maquette, synthetic protein scaffold H10A24 [?CH(3)()CONH-CGGGELWKL.HEELLKK.FEELLKL.AEERLKK. L-CONH(2)()?(2)](2). Removal of the iron porphyrin macrocycle from the high dielectric aqueous environment and sequestration within the hydrophobic core of the H10A24 maquette raises the equilibrium reduction midpoint potential by 36-138 mV depending on the hydrophobicity of the metalloporphyrin structure. By incorporating various natural and synthetic metalloporphyrins into a single protein scaffold, we demonstrate a 300-mV range in reduction potential modulation due to the electron-donating/withdrawing character of the peripheral macrocycle substituents. Solution pH is used to modulate the metalloporphyrin reduction potential by 160 mV, regardless of the macrocycle architecture, by controlling the protonation state of the glutamate involved in partial charge compensation of the ferric heme. Attempts to control the reduction potential by inserting charged amino acids into the hydrophobic core at close proximity to the metalloporphyrin lead to varied success, with H10A24-L13E lowering the E(m8.5) by 40 mV, H10A24-E11Q raising it by 50 mV, and H10A24-L13R remaining surprisingly unaltered. Modifying the charge of the adjacent metalloporphyrin, +1 for iron(III) protoporphyrin IX or neutral for zinc(II) protoporphyrin IX resulted in a loss of 70 mV [Fe(III)PPIX](+) - [Fe(III)PPIX](+) interaction observed in maquettes. Using these factors in combination, we illustrate a 435-mV variation of the metalloporphyrin reduction midpoint potential in a simple heme maquette relative to the about 800-mV range observed for natural cytochromes. Comparison between the reduction potentials of the heme maquettes and other de novo designed heme proteins reveals global trends in the E(m) values of synthetic cytochromes.     

FhuF, part of a siderophore-reductase system

FhuF is a cytoplasmic 2Fe-2S protein of Escherichia coli loosely associated with the cytoplasmic membrane. E. coli fhuF mutants showed reduced growth on plates with ferrioxamine B as the sole iron source, although siderophore uptake was not defective in transport experiments. Removal of iron from coprogen, ferrichrome, and ferrioxamine B was significantly lower in fhuF mutants compared to the corresponding parental strains, which suggested that FhuF is involved in iron removal from these hydroxamate-type siderophores. A redox potential E(1/2) of -310 +/- 25 mV relative to the normal hydrogen electrode was determined for FhuF by EPR redox titration; this redox potential is sufficient to reduce the siderophores coprogen and ferrichrome. M?ssbauer spectra revealed that FhuF in its [Fe(2+)-Fe(3+)] state is also capable of direct reduction of ferrioxamine B-bound ferric iron, thus proving its reductase function. This is the first report on a bacterial siderophore-iron reductase which in vivo seems to be specific for a certain group of hydroxamates.     

H2-Uptake Activity, Spectra, Reduction Potentials, and Kinetics of Iron Release on the Surface of Iron Core from Azotobacter vinelandii Bacterial Ferritin

Bacterial ferritin from Azotobacter vinelandii (AvBF_o has a function in H₂ uptake. The Fe³⁺ reduction on the surface of the iron core from AvBF_o is accompanied simultaneously by H₂ uptake, with a maximum activity of H₂ uptake of 450 H₂/AvBF_o. A reduction potential of ?402 mV for iron reduction on the surface of the core is found. A shift to the red the protein absorbance peaks ranging from 280 to 290 nm is observed between pH5 and 9 under 100% H₂ reduction. The reduction potential for iron release becomes negative at a rate of 0.025 mV/Fe²⁺ released. The kinetics of iron release on the surface of the core is a first-order reaction.     

H2-Uptake Activity, Spectra, Reduction Potentials, and Kinetics of Iron Release on the Surface of Iron Core from Azotobacter vinelandii Bacterial Ferritin

Bacterial ferritin from Azotobacter vinelandii (AvBF_o has a function in H₂ uptake. The Fe³⁺ reduction on the surface of the iron core from AvBF_o is accompanied simultaneously by H₂ uptake, with a maximum activity of H₂ uptake of 450 H₂/AvBF_o. A reduction potential of –402 mV for iron reduction on the surface of the core is found. A shift to the red the protein absorbance peaks ranging from 280 to 290 nm is observed between pH5 and 9 under 100% H₂ reduction. The reduction potential for iron release becomes negative at a rate of 0.025 mV/Fe²⁺ released. The kinetics of iron release on the surface of the core is a first-order reaction.     

Ennoblement of Stainless Steel by the Manganese-Depositing Bacterium Leptothrix discophora

The noble shift in open-circuit potential exhibited by microbially colonized stainless steel (ennoblement) was investigated by examining the relationship among surface colonization, manganese deposition, and open-circuit potential for stainless steel coupons exposed to batch cultures of the manganese-depositing bacterium Leptothrix discophora. Open-circuit potential shifted from -100 to +330 mV(infSCE) as a biofilm containing 75 nmol of MnO(infx) cm(sup-2) formed on the coupon surface but changed little further with continued MnO(infx) deposition up to 270 nmol cm(sup-2). Increased open-circuit potential corresponded to decreasing Mn(II) concentration in solution and to increased MnO(infx) accumulation and attached cell density on the coupon surfaces. MnO(infx) deposition was attributable to biological activity, and Mn(II) was observed to enhance cell attachment. The experimental results support a mechanism of ennoblement in which open-circuit potential is fixed near +350 mV(infSCE) by the cathodic activity of biomineralized MnO(infx).     

Determination of the redox properties of the Rieske [2Fe-2S] cluster of bovine heart bc1 complex by direct electrochemistry of a water-soluble fragment.

The redox potential of the Rieske [2Fe-2S] cluster of the bc1 complex from bovine heart mitochondria was determined by cyclic voltammetry of a water-soluble fragment of the iron/sulfur protein. At the nitric-acid-treated bare glassy-carbon electrode, the fragment gave an immediate and stable quasireversible response. The midpoint potential at pH 7.2, 25 degrees C and I of 0.01 M was Em = +312 +/- 3 mV. This value corresponds within 20 mV to results of an EPR-monitored dye-mediated redox titration. With increasing ionic strength, the midpoint potential decreased linearly with square root of I up to I = 2.5 M. From the cathodic-to-anodic peak separation, the heterogeneous rate constant, k degrees, was calculated to be approximately 2 x 10(-3) cm/s at low ionic strength; the rate constant increased with increasing ionic strength. From the temperature dependence of the midpoint potential, the standard reaction entropy was calculated as delta S degrees = -155 J.K-1.mol-1. The pH dependence of the midpoint potential was followed over pH 5.5-10. Above pH 7, redox-state-dependent pK changes were observed. The slope of the curve, -120 mV/pH above pH9, indicated two deprotonations of the oxidized protein. The pKa values of the oxidized protein, obtained by curve fitting, were 7.6 and 9.2, respectively. A group with a pKa,ox of approximately 7.5 could also be observed in the optical spectrum of the oxidized protein. Redox-dependent pK values of the iron/sulfur protein are considered to be essential for semiquinone oxidation at the Qo center of the bc1 complex.     

Zeta potential of synthetic and biological skin substitutes: effects on initial adherence

Early adherence of a skin substitute to the wound surface is paramount if it is to function as a skin equivalent. A surface electrical property (the zeta potential) was evaluated, and a positive correlation was found in which 5-hour adherence properties increased as the zeta potential became more positive. The following materials were tested: nylon-silicone composite (Z = -24.8 mV), Biobrane (Z = -15.2 mV), fresh-frozen porcine skin (Z = +12.5 mV), Opsite (Z = +14.9 mV), human amnion (Z = +18.2 mV), and human skin (Z = +23.0 mV). This order was also followed for increasing adherence values at 5 hours, which ranged from a low of 48.9 gm/cm2 for the nylon-silicone composite to a high of 88 gm/cm2 for human skin. Also determined was that both adherence and zeta potential decreased as increasing concentrations of glutaraldehyde were used to cross-link fresh-frozen porcine skin. Values ranged from a maximum of 85.5 gm/cm2 (0% glutaraldehyde; Z = +12.5 mV) to a minimum of 42.5 gm/cm2 (10% glutaraldehyde; Z = -26.4 mV). Additionally, deliberate biochemical modifications of porcine skin were undertaken in an attempt to increase zeta potential and adherence.     

Superoxide reductase from Desulfoarculus baarsii: identification of protonation steps in the enzymatic mechanism

Superoxide reductase (SOR) is a metalloenzyme that catalyzes the reduction of O2*- to H2O2 and provides an antioxidant mechanism in some anaerobic and microaerophilic bacteria. Its active site contains an unusual mononuclear ferrous center (center II). Protonation processes are essential for the reaction catalyzed by SOR, since two protons are required for the formation of H2O2. We have investigated the acido-basic and pH dependence of the redox properties of the active site of SOR from Desulfoarculus baarsii, both in the absence and in the presence of O2*-. In the absence of O2*-, the reduction potential and the absorption spectrum of the iron center II exhibit a pH transition. This is consistent with the presence of a base (BH) in close proximity to the iron center which modulates its reduction properties. Studies of mutants of the closest charged residues to the iron center II (E47A and K48I) show that neither of these residues are the base responsible for the pH transitions. However, they both interact with this base and modulate its pKa value. By pulse radiolysis, we confirm that the reaction of SOR with O2*- involves two reaction intermediates that were characterized by their absorption spectra. The precise step of the catalytic cycle in which one protonation takes place was identified. The formation of the first reaction intermediate, from a bimolecular reaction of SOR with O2*-, does not involve proton transfer as a rate-limiting step, since the rate constant k1 does not vary between pH 5 and pH 9.5. On the other hand, the rate constant k2 for the formation of the second reaction intermediate is proportional to the H+ concentration in solution, suggesting that the proton arises directly from the solvent. In fact, BH, E47, and K48 have no role in this step. This is consistent with the first intermediate being an iron(III)-peroxo species and the second one being an iron(III)-hydroperoxo species. We propose that BH may be involved in the second protonation process corresponding to the release of H2O2 from the iron(III)-hydroperoxo species.     

Modulation of redox potential and alteration in reactivity via the peroxide shunt pathway by mutation of cytochrome P450 around the proximal heme ligand

In the thermophilic cytochrome P450 from the thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 (P450st), a phenylalanine residue at position 310 and an alanine residue at position 320 are located close to the heme thiolate ligand, Cys317. Single site-directed mutants F310A and A320Q and double mutant F310A/A320Q have been constructed. All mutant enzymes as well as wild-type (WT) P450st were expressed at high levels. The substitution of F310 with Ala and of A320 with Gln induced shifts in redox potential and blue shifts in Soret absorption of ferrous-CO forms, while spectral characterization showed that in the resting state, the mutants almost retained the structural integrity of the active site. The redox potential of the heme varied as follows: -481 mV (WT), -477 mV (A320Q), -453 mV (F310A), and -450 mV (F310A/A320Q). The trend in the Soret band of the ferrous-CO form was as follows: 450 nm (WT) < 449 nm (A320Q) < 446 nm (F310A) < 444 nm (F310A/A320Q). These results established that the reduction potential and electron density on the heme iron are modulated by the Phe310 and Ala320 residues in P450st. The electron density on the heme decreases in the following order: WT > A320Q > F310A > F310A/A320Q. The electron density on the heme iron infers an essential role in P450 activity. The decrease in electron density interferes with the formation of a high-valent oxo-ferryl species called Compound I. However, steady-state turnover rates of styrene epoxidation with H2O2 show the following trend: WT approximately equal to A320Q < F310A approximately equal to F310A/A320Q. The shunt pathway which can provide the two electrons and oxygen required for a P450 reaction instead of NAD(P)H and dioxygen can rule out the first and second heme reduction in the catalytic process. Because the electron density on the heme iron might be deeply involved in the k cat values in this system, the intermediate Compound 0 which is the precursor species of Compound I mainly appears to participate dominantly in epoxidation with H2O2.     

Non-transferrin-bound iron uptake by rat liver. Role of membrane potential difference

Non-transferrin-bound iron is efficiently cleared from serum by the liver and may be primarily responsible for the hepatic damage seen in iron-overload states. We tested the hypothesis that transport of ionic iron is driven by the negative electrical potential difference across the liver cell membrane. Extraction of 55Fe-labeled ferrous iron (1 microM) from Krebs bicarbonate buffer by the perfused rat liver was continuously monitored as the transmembrane potential difference (measured using conventional microelectrodes) was altered over the physiologic range by isosmotic ion substitution. Resting membrane potential in Krebs bicarbonate buffer was -28 +/- 1 mV. Perfusion with 1 microM ferrous iron caused a reversible 3 +/- 1 mV depolarization, and higher concentrations of iron caused even greater depolarization. Conversely, depolarization of the liver cells consistently reduced iron extraction. Replacement of sodium with potassium (70 mM) or choline (131 mM) depolarized the hepatocytes to -15 and -20 mV and decreased iron extraction by 28 and 31%, respectively. Perfusion with bicarbonate-free solutions containing tricine buffer (10 mM) reduced the membrane potential to -23 mV and reduced iron extraction by 18%. In contrast, the high basal extraction of iron (91.1 +/- 1.4%) was not further increased by substitution of nitrate for chloride (-46 mV) or infusion of glucagon (-34 mV). All effects were reversible, suggesting that perfusion with 1 microM iron produced little toxicity. These findings are consistent with an electrogenic transport mechanism for uptake of non-transferrin-bound iron that is driven by the transmembrane potential difference.     

Redox potentials of the oriented film of the wild-type,the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins

The redox potentials of the oriented films of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins (bR), prepared by adsorbing purple membrane (PM) sheets or its mutant on a Pt electrode, have been examined. The redox potentials (V) of the wild-type bR were -470 mV for the 13-cis configuration of the retinal Shiff base in bR and -757 mV for the all-trans configuration in H(2)O, and -433 mV for the 13-cis configuration and -742 mV for the all-trans configuration in D(2)O. The solvent isotope effect (DeltaV=V(D(2)O)-V(H(2)O)), which shifts the redox potential to a higher value, originates from the cooperative rearrangements of the extensively hydrogen-bonded water molecules around the protonated C=N part in the retinal Schiff base. The redox potential of bR was much higher for the 13-cis configuration than that for the all-trans configuration. The redox potentials for the E194Q mutant in the extracellular region were -507 mV for the 13-cis configuration and -788 mV for the all-trans configuration; and for the E204Q mutant they were -491 mV for the 13-cis configuration and -769 mV for the all-trans configuration. Replacement of the Glu(194) or Glu(204) residues by Gln weakened the electron withdrawing interaction to the protonated C=N bond in the retinal Schiff base. The E204 residue is less linked with the hydrogen-bonded network of the proton release pathway compared with E194. The redox potentials of the D96N mutant in the cytoplasmic region were -471 mV for the 13-cis configuration and -760 mV for the all-trans configuration which were virtually the same as those of the wild-type bR, indicating that the D to N point mutation of the 96 residue had no influence on the interaction between the D96 residue and the C=N part in the Schiff base under the light-adapted condition. The results suggest that the redox potential of bR is closely correlated to the hydrogen-bonded network spanning from the retinal Schiff base to the extracellular surface of bR in the proton transfer pathway.