Structures of the Mo(V) forms of sulfite oxidase from Arabidopsis thaliana by pulsed EPR spectroscopy

The Mo(V) center of plant sulfite oxidase from Arabidopsis thaliana (At-SO) has been studied by continuous wave and pulsed EPR methods. Three different Mo(V) EPR signals have been observed, depending on pH and the technique used to generate the Mo(V) oxidation state. At pH 6, reduction by sulfite followed by partial reoxidation with ferricyanide generates an EPR spectrum with g-values similar to the low-pH (lpH) form of vertebrate SOs, but no nearby exchangeable protons can be detected. On the other hand, reduction of At-SO with Ti(III) citrate at pH 6 generates a Mo(V) signal with large hyperfine splittings from a single exchangeable proton, as is typically observed for lpH SO from vertebrates. Reduction of At-SO with sulfite at high pH generates the well-known high-pH (hpH) signal common to all sulfite oxidizing enzymes. It is proposed that, depending on the conformation of Arg374, the active site of At-SO may be in "closed" or "open" forms that differ in the degree of accessibility of the Mo center to substrate and water molecules. It is suggested that at low pH the sulfite-reduced At-SO has coordinated sulfate and is in the "closed form". Reoxidation to Mo(V) by ferricyanide leaves bound sulfate trapped at the active site, and consequently, there are no ligands with exchangeable protons. Reduction with Ti(III) citrate injects an electron directly into the active site to generate the [Mo(V)[triple bond]O(OH)]2+ unit that is well-known from model chemistry and which has a single exchangeable proton with a large isotropic hyperfine interaction. At high pH, the active site is in the "open form", and water can readily exchange into the site to generate the hpH SO.     

Spectroscopic studies of the molybdenum-containing dimethyl sulfoxide reductase from Rhodobacter sphaeroides f. sp. denitrificans.

Absorption and EPR spectroscopic properties of purified dimethyl sulfoxide (Me2SO) reductase from Rhodobacter sphaeroides f. sp. denitrificans have been examined. The absence of prosthetic groups other than the molybdenum center in the enzyme has made it possible to study its absorption properties. The enzyme displays multiple absorbance peaks in both the oxidized and the dithionite-reduced forms. The oxidized enzyme has absorbance peaks at 280, 350, 470, 550, and 720 nm while the dithionite-reduced enzyme has peaks at 280, 374, and 645 nm with a shoulder at 430 nm. A comparison of the absorbance spectrum of oxidized Me2SO reductase with that of the molybdenum fragment of rat liver sulfite oxidase shows that the 350 and 470 peaks are common to both proteins. EPR studies of the Mo(V) form of Me2SO reductase show a rhombic signal with g1 = 1.988, g2 = 1.977, g3 = 1.961, and g(ave) = 1.975. The signal shows evidence of coupling to an exchangeable proton with A1 = 1.05, A2 = 1.13, A3 = 0.98, and Aave = 1.05 millitesla. These parameters are similar to those of other Mo enzymes, however, the epr signal of this enzyme differs from those of other Mo hydroxylases in showing only a slight sensitivity to pH and no detectable anion effect. EPR potentiometric titrations of Me2SO reductase gave midpoint potentials of +144 mV for the Mo(VI)/Mo(V) couple and +160 mV for the Mo(V)/Mo(IV) couple at room temperature and +141 mV for the Mo(VI)/Mo(V) couple and +200 mV for the Mo(V)/Mo(IV) couple at 173 K.     

Circular dichroism and potentiometry of FAD, heme and Mo-pterin prosthetic groups of assimilatory nitrate reductase

Oxidation-reduction midpoint potentials for flavin, heme, and molybdenum-pterin prosthetic groups of assimilatory nitrate reductase (NR) from Chlorella vulgaris were measured at room temperature by using CD and EPR potentiometry. The CD changes accompanying reduction of each prosthetic group were determined by using enzyme fragments containing either FAD or heme and molybdenum prosthetic groups, obtained by limited proteolysis, and by poising the enzyme at various redox potentials in the presence of dye mediators. Limited proteolysis did not appear to alter the environment of the prosthetic groups, as judged by their CD spectra. Also, CD potentiometric titration of FAD in intact NR (Em' = -272 mV, n = 2) gave a similar value (Em' = -286 mV) to the FAD of the flavin-containing proteolytic domain, determined by visible spectroscopy. Less than 1% of the flavin semiquinone was detected by EPR spectroscopy, indicating that Em' (FAD/FAD.-) may be more than 200 mV lower than Em' (FAD.-/FADH-). Reduction of heme resulted in splitting of both Soret and alpha CD bands into couplets. The heme Em' was -162 mV (n = 1) determined by both CD and visible spectroscopy. Reduction of Mo-pterin was followed by CD at 333 nm, and Mo(V) was monitored by room temperature EPR spectroscopy. Most of the change in the Mo-pterin CD spectrum was due to the Mo(VI)/Mo(V) transition. The Em' values determined for Mo(VI)/Mo(V) were +26 mV by CD and +16 mV by EPR, whereas Mo(V)/Mo(IV) values were -40 mV by CD and -26 mV by EPR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrochemical studies of arsenite oxidase: an unusual example of a highly cooperative two-electron molybdenum center

Arsenite oxidase from Alcaligenes faecalis, an unusual molybdoenzyme that does not exhibit a Mo(V) EPR signal during oxidative-reductive titrations, has been investigated by protein film voltammetry. A film of the enzyme on a pyrolytic graphite edge electrode produces a sharp two-electron signal associated with reversible reduction of the oxidized Mo(VI) molybdenum center to Mo(IV). That reduction or oxidation of the active site occurs without accumulation of Mo(V) is consistent with the failure to observe a Mo(V) EPR signal for the enzyme under a variety of conditions and is indicative of an obligate two-electron center. The reduction potential for the molybdenum center, 292 mV (vs SHE) at pH 5.9 and 0 degrees C, exhibits a linear pH dependence for pH 5-10, consistent with a two-electron reduction strongly coupled to the uptake of two protons without a pK in this range. This suggests that the oxidized enzyme is best characterized as having an L(2)MoO(2) rather than L(2)MoO(OH) center in the oxidized state and that arsenite oxidase uses a "spectator oxo" effect to facilitate the oxo transfer reaction. The onset of the catalytic wave observed in the presence of substrate correlates well with the Mo(VI/IV) potential, consistent with catalytic electron transport that is limited only by turnover at the active site. The one-electron peaks for the iron-sulfur centers are difficult to observe by protein film voltammetry, but spectrophotometric titrations have been carried out to measure their reduction potentials: at pH 6.0 and 20 degrees C, that of the [3Fe-4S] center is approximately 260 mV and that of the Rieske center is approximately 130 mV.     

Oxidation-reduction potentials of flavin and Mo-pterin centers in assimilatory nitrate reductase: variation with pH

Potentiometric titrations of assimilatory nitrate reductase from Chlorella vulgaris were performed within the pH range 6.0-9.0. Mo(V) was measured by room temperature EPR spectroscopy while the reduction state of FAD was monitored by CD spectroscopy. Between pH 6 and 8.5, the line shape of the Mo(V) EPR signal was constant, exhibiting superhyperfine coupling to a single, exchangeable proton. Potentiometric titrations indicated the Em values for the Mo(VI)/Mo(V) (+61 mV, pH 6) and Mo(V)/Mo(IV) (+35 mV, pH 6) couples decreased with increasing pH by approximately -59 mV/pH unit, consistent with the uptake of a single proton upon reduction of Mo(VI) to Mo(V) and Mo(V) to Mo(IV). The pKa values for the dissociation of these redox-coupled protons appeared to lie outside the pH range studied: pKo(MoVI), pKo(MoV) less than 5.5; pKr(MoV), pKr(MoIV) greater than 9. The Em (n = 2) for FAD (-250 mV, pH 7) varied by approximately -30 mV/pH unit within the pH range 6.0-9.0. Low-temperature EPR potentiometry at the extreme pH values indicated less than 0.5% conversion of FAD to the semiquinone form at the midpoint of the titrations. In contrast, NADH-reduced enzyme exhibited approximately 3-5% of the FAD in the semiquinone form, present as the anionic (FAD.-) species, the spectrum characterized by a line width of 1.3 mT at both pH 6.0 and 9.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidase reaction of cytochrome cd(1) from Paracoccus pantotrophus

Cytochrome cd(1) (cd(1)NIR) from Paracoccus pantotrophus, which is both a nitrite reductase and an oxidase, was reduced by ascorbate plus hexaamineruthenium(III) chloride on a relatively slow time scale (hours required for complete reduction). Visible absorption spectroscopy showed that mixing of ascorbate-reduced enzyme with oxygen at pH = 6.0 resulted in the rapid oxidation of both types of heme center in the enzyme with a linear dependence on oxygen concentration. Subsequent changes on a longer time scale reflected the formation and decay of partially reduced oxygen species bound to the d(1) heme iron. Parallel freeze-quench experiments allowed the X-band electron paramagnetic resonance (EPR) spectrum of the enzyme to be recorded at various times after mixing with oxygen. On the same millisecond time scale that simultaneous oxidation of both heme centers was seen in the optical experiments, two new EPR signals were observed. Both of these are assigned to oxidized heme c and resemble signals from the cytochrome c domain of a "semi-apo" form of the enzyme for which histidine/methionine coordination was demonstrated spectroscopically. These observations suggests that structural changes take around the heme c center that lead to either histidine/methionine axial ligation or a different stereochemistry of bis-histidine axial ligation than that found in the as prepared enzyme. At this stage in the reaction no EPR signal could be ascribed to Fe(III) d(1) heme. Rather, a radical species, which is tentatively assigned to an amino acid radical proximal to the d(1) heme iron in the Fe(IV)-oxo state, was seen. The kinetics of decay of this radical species match the generation of a new form of the Fe(III) d(1) heme, probably representing an OH(-)-bound species. This sequence of events is interpreted in terms of a concerted two-electron reduction of oxygen to bound peroxide, which is immediately cleaved to yield water and an Fe(IV)-oxo species plus the radical. Two electrons from ascorbate are subsequently transferred to the d(1) heme active site via heme c to reduce both the radical and the Fe(IV)-oxo species to Fe(III)-OH(-) for completion of a catalytic cycle.     

Solution equilibrium studies on metal complexes of 2,3-dihydroxy-phenylalanine-hydroxamic acid (Dopaha) and models: catecholate versus hydroxamate coordination in iron(III)-, aluminium(III)- and molybdenum(VI)-Dopaha complexes

Equilibrium results based on pH potentiometric, spectrophotometric and (1)H NMR measurements for the complexes of Fe(III), Al(III) and Mo(VI) with 2,3-dihydroxy-phenylalanine-hydroxamic acid (Dopaha) as well as for binary model systems Fe(III)-, Al(III)-, Mo(VI)-acetohydroxamic acid (Aha), -alpha-alaninehydroxamic acid (alpha-Alaha) and -1,2-dihydroxy-3,5-benzene-disulphonate (Tiron) and ternary model systems Fe(III)-, Al(III)-, Mo(VI)-Tiron-Aha, are summarized in this paper. The amine-type coordination mode is not detectable with these metal ions at all. Precipitation occurs at pH <5.5 with Fe(III) and Al(III) even at a Dopaha-to-metal ion ratio of 10:1. Hydroxamate-type coordination was demonstrated with both metals below the pH range of precipitation but, after dissolution, catecholate-type coordination was exclusively found. The hydroxamate-type coordination mode occurs only in the very acidic pH range for Mo(VI) complexes and the crossover from hydroxamate to catecholate binding occurs at pH >3. A ligand-bridged dinuclear species, [(MoO(2))(2)(Dopaha)(2)](2+), involving mixed-type (catecholate and hydroxamate) coordination modes is formed in the pH range 2.5-5.5. [MoO(2)A(2)H(2)], with catecholate-type coordination, forms above pH 3. On increasing the pH further, deprotonation of the coordinated Dopaha and hydrolytic processes result in the formation of catecholate-coordinated [MoO(3)AH] and [MoO(3)A]. MoO(4)(2-) and free Dopaha exist above pH 10.     

Oxidation-reduction midpoint potentials of the molybdenum center in spinach NADH:nitrate reductase

Oxidation-reduction midpoint potentials for the molybdenum center in assimilatory NADH:nitrate reductase isolated from spinach (Spinacia oleracea) have been determined at pH 7.0 in the presence of dye mediators using EPR spectroscopy to monitor formation of Mo(V). Values for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples were determined to be -8 and -42 mV, respectively.     

The heterogeneity of arginases in rat tissues.

1. The mid-point reduction potentials of the various groups in xanthine oxidase from bovine milk were determined by potentiometric titration with dithionite in the presence of dye mediators, removing samples for quantification of the reduced species by e.p.r. (electron-paramagnetic-resonance) spectroscopy. The values obtained for the functional enzyme in pyrophosphate buffer, pH8.2, are: Fe/S centre I, -343 +/- 15mV; Fe/S II, -303 +/- 15mV; FAD/FADH-; -351 +/- 20mV; FADH/FADH2, -236 +/-mV; Mo(VI)/Mo(V) (Rapid), -355 +/- 20mV; Mo(V) (Rapid)/Mo(IV), -355 +/- 20mV. 2. Behaviour of the functional enzyme is essentially ideal in Tris but less so in pyrophosphate. In Tris, the potential for Mo(VI)/Mo(V) (Rapid) is lowered relative to that in pyrophosphate, but the potential for Fe/S II is raised. The influence of buffer on the potentials was investigated by partial-reduction experiments with six other buffers. 3. Conversion of the enzyme with cyanide into the non-functional form, which gives the Slow molybdenum signal, or alkylation of FAD, has little effect on the mid-point potentials of the other centres. The potentials associated with the Slow signal are: Mo(VI)/Mo(V) (Slow), -440 +/- 25mV; Mo(V) (Slow)/Mo(IV), -480 +/- 25 mV. This signal exhibits very sluggish equilibration with the mediator system. 4. The deviations from ideal behaviour are discussed in terms of possible binding of buffer ions or anti-co-operative interactions amongst the redox centres.     

Electron paramagnetic resonance properties and oxidation-reduction potentials of the molybdenum,flavin, and iron-sulfur centers of chicken liver xanthine dehydrogenase

The oxidation-reduction potentials of the various prosthetic groups in the native and desulfo forms of chicken liver xanthine dehydrogenase, determined by potentiometric titration in 0.05 m potassium phosphate buffer, pH 7.8, are: Mo(VI)/Mo(V) (native), ?357 mV; Mo(VI)/Mo(V) (desulfo), ?397 mV; Mo(V)/Mo(IV) (native), ?337 mV; Mo(V)/Mo(IV) (desulfo), ?433 mV; FAD/FADH · ?345 mV; FADH · FADH₂, ? 377 mV; (Fe/S)I_ox/(Fe/S)I_red, ?280 mV; (Fe/S)II_ox/(Fe/S)II_red, ? 275 mV. Titration at pH 6.8 revealed that the Mo and FAD centers but not the Fe/S centers are in prototropic equilibrium. Spectroscopic studies on the native and deflavinated enzymes show that environment of the flavin in xanthine dehydrogenase differs from that in bovine milk xanthine oxidase.     

Electron paramagnetic resonance of the tungsten derivative of rat liver sulfite oxidase.

Sulfite oxidase purified from livers of tungsten-treated rats has been used for EPR studies of tungsten substituted at the molybdenum site of the enzyme in a fraction of the molecules. The EPR signal of W(V) in sulfite oxidase is quite similar to that of Mo(V) in its line shape and in its sensitivity to the presence of anions such as phosphate and fluoride. Hyperfine interaction with a dissociable proton is also observed in both signals. The pH-dependent alteration in line shape exhibited by the Mo(V) EPR signal of the rat liver enzyme. Incomplete reduction of the tungsten center at pH 9 is indicated by attenuated signal intensity at this pH. The W(V) signal has g values lower than those of the Mo(V) signal, has a much broader resonance envelope, and is much less readily saturated by increasing microwave power. Kinetic studies on the reduction of the heme and tungsten centers of sulfite oxidase have shown that reduction of de-molybdo forms of sulfite oxidase by sulfite is catalyzed by the residual traces of native molybdenum-containing molecules. Reduction is accomplished by electron transfer involving intermolecular heme-heme interaction. The W(V) signal is generated only after all the heme centers are reduced. The rate and extent of heme reduction at pH 9 are the same as at pH 7. Studies on the reoxidation of W(V) and reduced heme by O2 and by cytochrome c suggest that the cytochrome b5 of sulfite oxidase is the site of electron transfer to cytochrome c, whereas oxidase activity is the property of the molybdenum center. It appears that the tungsten center in sulfite oxidase is incapable of oxidizing sulfite.     

Bacterial Reduction of Cr(VI) and Fe(III) in In Vitro Conditions

ABSTRACT Chemical reduction of Cr(VI) can be a strategy to detoxify toxic metals in oxidized states, whereas reduction of Fe(III) could enhance the availability of Fe in the form of Fe(II) to boost plant growth. However, it creates another problem of chemical sludge disposal. Hence, microbial conversion of Cr(VI) to Cr(III) and Fe(III) to Fe(II) is preferred over the chemical method. Out of 11 bacterial strains isolated from the rhizospheric zone of Typha latifolia growing on fly ash dump sites, four isolates were selected for the reduction of Cr(VI) and Fe(III) and were identified as Micrococcus roseus NBRFT2 (MTCC 9018), Bacillus endophyticus NBRFT4 (MTCC 9021), Paenibacillus macerans NBRFT5 (MTCC 8912), and Bacillus pumilus NBRFT9 (MTCC 8913). These strains were individually tested for survival at different concentrations of Cr(VI) and Fe(III), pH, and temperature, and then, their ability for reduction of both metals was evaluated at optimum pH 8.0 and temperature 35°C. The results indicated that NBRFT5 was able to reduce the maximum amount, 99% Cr(VI) and 98% Fe(III). Other strains also reduced these metals to different levels, but less than NBRFT5. Hence, these strains may be used for decontamination of metal-contaminated sites, particularly with Cr(VI) and Fe(III) through the reduction process.     

异化铁还原细菌LQ25还原重金属Cr (VI)的特性研究

[背景] 异化铁还原细菌能够在还原Fe （III）的同时将毒性较大的Cr （VI）还原成毒性较小的Cr （III）,解决铬污染的问题。[目的] 基于丁酸梭菌（Clostridium butyricum） LQ25异化铁还原过程制备生物磁铁矿,开展异化铁还原细菌还原Cr （VI）的特性研究。[方法] 构建以氢氧化铁为电子受体和葡萄糖为电子供体的异化铁培养体系。菌株LQ25培养结束时制备生物磁铁矿。设置不同初始Cr （VI）浓度（5、10、15、25和30 mg/L）,分别测定菌株LQ25对Cr （VI）还原效率以及生物磁铁矿对Cr （VI）的还原效率。[结果] 菌株LQ25在设置的Cr （VI）浓度范围内都能良好生长。当Cr （VI）浓度为15 mg/L时,在异化铁培养条件下,菌株LQ25对Cr （VI）的还原率为63.45%±5.13%,生物磁铁矿对Cr （VI）的还原率为87.73%±9.12%,相比菌株还原Cr （VI）的效率提高38%。pH变化能影响生物磁铁矿对Cr （VI）的还原率,当pH 2.0时,生物磁铁矿对Cr （VI）的还原率最高,几乎达到100%。电子显微镜观察发现生物磁铁矿表面有许多孔隙,X-射线衍射图谱显示生物磁铁矿中Fe （II）的存在形式是Fe （OH）₂。[结论] 基于异化铁还原细菌制备生物磁铁矿可用于还原Cr （VI）,这是一种有效去除Cr （VI）的途径。     

Studies of the Mo(V) Center of the Y343F Mutant of Human Sulfite Oxidase by Variable Frequency Pulsed EPR Spectroscopy

The Mo(V) forms of the Tyr343Phe (Y343F) mutant of human sulfite oxidase (SO) have been investigated by continuous wave (CW) and variable frequency pulsed EPR spectroscopies as a function of pH. The CW EPR spectrum recorded at low-pH (∼6.9) has g-values similar to those known for the low-pH form of the native vertebrate SO (original lpH form); however, unlike the spectrum of original lpH SO, it does not show any hyperfine splittings from a nearby exchangeable proton. The detailed electron spin echo (ESE) envelope modulation (ESEEM) and pulsed electron-nuclear double resonance (ENDOR) experiments also did not reveal any nearby protons that could belong to an exchangeable ligand at the molybdenum center. These results suggest that under low-pH conditions the active site of Y343F SO is in the “blocked” form, with the Mo(V) center coordinated by sulfate. With increasing pH the EPR signal from the “blocked” form decreases, while a signal similar to that of the original lpH form appears and becomes the dominant signal at pH >9. In addition, both the CW EPR and ESE-detected field-sweep spectra reveal a considerable contribution from a signal similar to that usually detected for the high-pH form of native vertebrate SO (original hpH form). The nearby exchangeable protons in both of the component forms observed at high-pH were studied by the ESEEM spectroscopy. These results indicate that the Y343F mutation increases the apparent pK_a of the transition from the lpH to hpH forms by ∼2 pH units.     

Formate dehydrogenase from Methanobacterium formicicum. Electron paramagnetic resonance spectroscopy of the molybdenum and iron-sulfur centers

Formate dehydrogenase from Methanobacterium formicicum was examined by electron paramagnetic resonance spectroscopy. Although oxidized enzyme yielded no EPR signals over the temperature range 8-200 K, dithionite reduction resulted in generation of two paramagnetic components. The first, a nearly isotropic signal visible at temperatures below 200 K with g1 = 2.018, g2 = 2.003, and g3 = 1.994, exhibited nuclear hyperfine interaction with two equivalent protons (A1 = 0.45, A2 = 0.6, and A3 = 0.55 milliTeslas). EPR spectra of partially reduced 95Mo-enriched formate dehydrogenase exhibited additional 3-4 milliTeslas splittings, due to spin interaction with the 95Mo nucleus. Thus, this signal is due to a Mo center. This is the first reported example of a Mo center with gav greater than 2.0 in a biological system. The second species, a rhombic signal visible below 40 K with g values of g1 = 2.0465, g2 = 1.9482, and g3 = 1.9111 showed no hyperfine coupling and was assigned to reduced Fe/S. Both paramagnetic species could be detected in samples of M. formicicum whole cells anaerobically reduced with sodium formate. The Mo(V) signal was altered following addition of cyanide (g1 = 1.996, g2 = 1.988, and g3 = 1.980). Growth of bacteria in the presence of 1 mM WO4(2-) resulted in abolition of the Mo(V) EPR signal and formate dehydrogenase activity. Em, 7.7 was -330 mV for Mo(VI)/Mo(V) and -470 mV for Mo(V)/Mo(IV).     

A manganese(IV)/iron(IV) intermediate in assembly of the manganese(IV)/iron(III) cofactor of Chlamydia trachomatis ribonucleotide reductase

We recently showed that the class Ic ribonucleotide reductase from the human pathogen Chlamydia trachomatis uses a Mn(IV)/Fe(III) cofactor to generate protein and substrate radicals in its catalytic mechanism [Jiang, W., Yun, D., Saleh, L., Barr, E. W., Xing, G., Hoffart, L. M., Maslak, M.-A., Krebs, C., and Bollinger, J. M., Jr. (2007) Science 316, 1188-1191]. Here, we have dissected the mechanism of formation of this novel heterobinuclear redox cofactor from the Mn(II)/Fe(II) cluster and O2. An intermediate with a g = 2 EPR signal that shows hyperfine coupling to both 55Mn and 57Fe accumulates almost quantitatively in a second-order reaction between O2 and the reduced R2 complex. The otherwise slow decay of the intermediate to the active Mn(IV)/Fe(III)-R2 complex is accelerated by the presence of the one-electron reductant, ascorbate, implying that the intermediate is more oxidized than Mn(IV)/Fe(III). M?ssbauer spectra show that the intermediate contains a high-spin Fe(IV) center. Its chemical and spectroscopic properties establish that the intermediate is a Mn(IV)/Fe(IV)-R2 complex with an S = 1/2 electronic ground state arising from antiferromagnetic coupling between the Mn(IV) (S(Mn) = 3/2) and high-spin Fe(IV) (S(Fe) = 2) sites.     

Redox potential of the active bleomycin-Fe(III) complex by cyclic voltammetry

The redox potential of the active Fe(III) complex of bleomycin (BLM), which is a DNA cleaving species, was measured by cyclic voltammetry at 25 °C under a hydrogen atmosphere. The cyclic voltammogram showed the reversible one-electron Fe(III)/Fe(II) coupled redox reaction at −0.225 V versus SCE. Under the same conditions the redox potentials of the iso-BLM—Fe(III) complex and the deglyco-BLM—Fe(III) complex were also observed, but the cyclic voltammogram for the inactive Fe(III) complex of BLM could not be obtained.     

Intramolecular photoredox reactions in iron(III) cytochrome c and its azide derivative

The irradiation of deaerated solutions of horse heart cytochrome c causes the reduction of Fe(III) to Fe(II). The dependence of the photoreaction quantum yield on pH shows that the photoreactive species is a form of cytochrome c which contains methionine-80 and histidine-18 as heme ligands. The primary photochemical event consists of an electron transfer from the sulphur of methionine- 80 to iron. The re-oxidation of the photochemically obtained Fe(II) protein gives a Fe(III) cytochrome which exhibits a typical low-spin absorption spectrum, lacking the 695-nm band and indicating that a strong field ligand, other than methionine-80, coordinates to the sixth binding site of the heme iron. Spectrophotometric titration of the photochemically modified Fe(III) cytochrome shows that histidine- 18 remains bound in the fifth position.The substitution of methionine-80 with the more oxidizable azide ligand increases the efficiency of the intramolecular electron transfer. Azide radicals, detected by spin-trapping ESR technique, are formed in the primary act. Visible-UV spectral data indicate that histidine-18 and methionine-80 occupy the fifth and sixth position, respectively, in the photoreaction product. All the results obtained correlate well with those previously obtained in investigations concerning the photoredox behavior of iron porphyrin complexes.     

Characterization of the reduction steps of Fe(III) porphyrins

Polarographic studies have shown that Fe(III) porphyrins undergo successively three one-electron reduction steps in dimethylformamide. The first involves the Fe(III)/Fe(II) redox couple. The second step proceeds to a second reduction of the metal ion and is attributed to the Fe(II)/Fe(I)_couple. This new reduction state of iron porphyrins has been characterized by ESR spectra and by absorption spectra in various solvents. This compound is not axially liganded by strong nucleophilic bases but is sensitive to solvation, the lone electron being localised in the d_z2 orbital. The third reduction step is assumed to involve a reduction of the porphyrin π-electron system.All these results have been confirmed by chemical reductions in tetrahydrofuran.     

Characterization of arsenite-complexed xanthine oxidase at room temperature. Spectral properties and pH-dependent redox behavior of the molybdenum-arsenite center

Several aspects of the interaction of xanthine oxidase with arsenite are investigated. Room temperature potentiometric titrations using EPR to monitor Molybdenum reduction reveal midpoint potentials of -225 mV for the Mo(VI)-arsenite/Mo(V)-arsenite couple and -440 mV for the Mo(V)-arsenite/Mo(IV)-arsenite couple at pH 8.3. Under the same conditions, the values for native enzyme are -395 mV and -420 mV, respectively. The predicted effects of the altered Mo(VI)/Mo(V) potential on the distributions of reducing equivalents in partially reduced enzyme are compared with the experimentally observed effects in optical experiments. The bleaching that occurs on reduction of the chromophore that is generated when arsenite binds to oxidized enzyme is characterized and found to be associated with reduction of Mo(V)-arsenite to Mo(V)-arsenite. This probe enables determination of the midpoint potential for this conversion using optical data. From such data at a series of pH values ranging from 6.15 to 9.9, a pH dependence of -60 mV/pH unit increase is determined for this couple above pH 7. The ability of arsenite to bind to reduced xanthine oxidase and to desulfo enzyme are also investigated. Reduced active enzyme binds arsenite much more tightly (Kd less than 0.1 microM) and more rapidly than does oxidized active enzyme (Kd = 8 microM); oxidized desulfo enzyme binds arsenite almost as tightly (Kd = 20 microM) as does the oxidized active enzyme.