Anaerobic iron deposition into horse spleen, recombinant human heavy and light and bacteria ferritins by large oxidants

Large-molecule oxidants oxidize Fe(II) to form Fe(III) cores in the interior of ferritins at rates comparable to or faster than the iron deposition reaction using O(2) as oxidant. Iron deposition into horse spleen ferritin (HoSF) occurs using ferricyanide ion, 2,6-dichlorophenol-indophenol, and several redox proteins: cytochrome c, stellacyanin, and ceruloplasmin. Cytochrome c also loads iron into recombinant human H-chain (rHF), human L-chain (rLF), and A. vinelandii bacterioferritin (AvBF). The enzymatic activities of ferritins were monitored anaerobically using stopped-flow kinetic spectrophotometry. The reactions exhibit saturation kinetics with respect to the large oxidant concentrations, giving apparent Michaelis constants for cytochrome c as oxidant: K(m)=39.6 microM for HoSF and 6.9 microM for AvBF. Comparison of the kinetic parameters with that of iron deposition by O(2) shows that large oxidants load iron into HoSF and AvBF more effectively than O(2) and may use a mechanism different than the ferroxidase center. Large oxidants did not deposit iron as efficiently with rHF and rLF. The results suggest that the heme groups in AvBF and the protein redox centers present in heteropolymers may assist in anaerobic iron deposition by large oxidants. The physiological relevance of iron deposition by large molecules, including protein oxidants is discussed.     

The oxidation of C-550 by exogenously added oxidants in the presence of dichlorophenyldimethylurea: The localization of the primary electron acceptor of Photosystem II in the thylakoid membrane

The oxidation of C-550 by exogenously added oxidants in spinachchloroplasts and digitonin-treated chloroplasts was studiedin the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inan attempt to elucidate localization of the primary electronacceptor of Photosystem II in the thylakoid membrane. C-550was directly oxidized by various oxidants such as ferricyanide,N-methylphenazonium methosulfate (PMS) and quinones with redoxpotentials higher than that of C-550. Among the oxidants used,dibromothymoquinone was the most effective oxidant of C-550,followed by PMS. In spite of its high redox potential, ferricyanidewas rather a poor oxidant. The rates of C-550 oxidation by quinoneswere linearly proportional to the oxidant concentration, whereasthe rates tapered off with increasing concentrations of polaroxidants within the ranges of concentration used. These resultsindicate that C-550 is located inside the thylakoid membraneand is covered by a lipophilic shield. Addition of cations,especially divalent cations, significantly affected C-550 oxidationby ferricyanide or PMS but not by nonpolar oxidants. C-550 oxidationby ferricyanide was accelerated at low pH. Thus the accessibilityof C-550 to polar oxidants appears to be affected by electrostaticinteractions between oxidant ions and the negative charges ofthe thylakoid surface. When electrostatic interaction was minimized,ferricyanide oxidized C-550 as rapidly as several quinones did.This suggests that C-550 is located close to the surface ofthe membrane. The evidence indicates that the rates of C-550oxidation depend not only on the accessibility of C-550 to addedoxidants, but also on the reactivity between them. Reduction of cytochrome f by added reductants shows featuressimilar to those of C-550 oxidation by added oxidants, indicatingthat properties of the shield covering cytochrome f are similarto those of the shield covering C-550. (Received March 8, 1977; )     

Electrostatic effects on the kinetics of electron transfer reactions of cytochrome c caused by binding to negatively charged lipid bilayer vesicles.

The effect of binding reduced tuna mitochondrial cytochrome c to negatively charged lipid bilayer vesicles at low ionic strength on the kinetics of electron transfer to various oxidants was studied by stopped-flow spectrophotometry. Binding strongly stimulated (up to 100-fold) the rate of reaction with the positively charged cobalt phenanthroline ion, whereas the rate of reaction with the negatively charged ferricyanide ion was greatly inhibited (up to 60-fold), as compared with the same systems either at high ionic strength or at low ionic strength either in the presence of electrically neutral vesicles or in the absence of vesicles. Reactions of tuna cytochrome c with uncharged or electrically neutral oxidants such as benzoquinone and Rhodospirillum rubrum cytochrome c2 were unaffected by binding to vesicles, suggesting little or no effect of membrane association on cytochrome structure or accessibility of the heme center. The kinetic effects were largest at lower cytochrome c to vesicle ratios, where there was a greater degree of exposure of negatively charged regions on the membrane. The reduction of cobalt phenanthroline and ferricyanide by bound cytochrome c proceeded by nonexponential kinetics, as compared with the monophasic kinetics observed in the absence of vesicles. This was probably due to the heterogeneous distribution of vesicle sizes which exists at a given lipid to protein ratio. Nonlinear oxidant concentration dependencies were observed for cobalt phenanthroline oxidation of membrane-bound cytochrome c, consistent with a (minimal) two-step kinetic mechanism involving association of the oxidant with the membrane followed by electron transfer. Based on a comparison of second-order rate constants as a function of lipid to protein mole ratio, binding of cytochrome c to the bilayer increased the efficiency of the cobalt phenanthroline reaction by a factor of approximately 500 at the highest lipid:protein ratio used. The results suggest a mechanism involving attractive and repulsive electrostatic interactions between the negatively charged bilayer and the electrically charged oxidants, which increase or decrease their effective concentrations at the membrane surface.     

Simultaneous photoreduction and photooxidation of cytochrome b-559 in Photosystem II treated with carbonylcyanide-m-chlorophenylhydrazone

The possibility of a Photosystem II (PS II) cyclic electron flow via Cyt b-559 catalyzed by carbonylcyanide m-chlorophenylhydrazone (CCCP) was further examined by studying the effects of the PS II electron acceptor 2,6-dichloro-p-benzoquinone (DCBQ) on the light-induced changes of the redox states of Cyt b-559. Addition to barley thylakoids of micromolar concentrations of DCBQ completely inhibited the changes of the absorbance difference corresponding to the photoreduction of Cyt b-559 observed either in the presence of 10 M ferricyanide or after Cyt b-559 photooxidation in the presence of 2 M CCCP. In CCCP-treated thylakoids, the concentration of photooxidized Cyt b-559 decreased as the irradiance of actinic light increased from 2 to 80 W m^-2 but remained close to the maximal concentration (0.53 photooxidized Cyt b-559 per photoactive Photosystem II) in the presence of 50 M DCBQ. The stimulation of Cyt b-559 photooxidation in parallel with the inhibition of its photoreduction caused by DCBQ demonstrate that the extent of the light-induced changes of the redox state of Cyt b-559 in the presence of CCCP is determined by the difference between the rates of photooxidation and photoreduction of Cyt b-559 occuring simultaneously in a cyclic electron flow around PS II.We also observed that the Photosystem I electron acceptor methyl viologen (MV) at a concentration of 1 mM barely affected the rate and extent of the light-induced redox changes of Cyt b-559 in the presence of either FeCN or CCCP. Under similar experimental conditions, MV strongly quenched Chl-a fluorescence, suggesting that Cyt b-559 is reduced directly on the reducing side of Photosystem II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting system Y - ANT-2p 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene - CCCP carbonylcyanide-m-chlorophenylhydrazone - DCBQ 2,6-dichloro-p-benzoquinone - FeCN ferricyanide - MV methyl viologen - P₆₈₀ Photosystem II reaction center Chl-a dimer CIW-DPB publication No. 1118.     

Effect of ionophores on carrier-mediated electron translocation in ferricyanide-containing liposomes

Ferricyanide-containing liposomes were used as a system to compare the electron- and proton-translocating properties of six redox reagents commonly used as electron donors for biochemical systems. The effects of different ionophore combinations on the ferricyanide-reduction rate were generally consistent with the expected proton- and electron-translocating properties of the mediators. The transmembrane pH gradient produced by hydrogen carriers was demonstrated. Nigericin or valinomycin plus carbonyl cyanide p-trifluoromethoxyphenylhydrazone are capable of collapsing this gradient and of stimulating ferricyanide reduction mediated by this type of carrier. No pH gradient is produced with the electron carrier 1,1'-dibutylferrocene. In the presence of tetraphenylboron anion, which is needed for this carrier to act as an efficient mediator, addition of valinomycin alone is sufficient to obtain full stimulation of ferricyanide reduction. NNN'N'-Tetramethyl-p-phenylenediamine does not behave as a simple electron carrier. During NNN'N'-tetramethyl-p-phenylenediamine-mediated ferricyanide reduction protons are translocated across the membrane and accumulated in the vesicles. This is not due to the presence of demethylated impurities in the NNN'N'-tetramethyl-p-phenylenediamine sample, but may be the result of an accumulation of oxidation products other than the Wurster's Blue radical. These results suggest a reconsideration of studies on protonmotive forces across membranes where NNN'N'-tetramethyl-p-phenylenediamine is used as a mediator.     

Novel redox surfactants and their interactions with glucose oxidase of Aspergillus niger

A number of novel redox surfactants (based on mixed bipyridine/dipyridylamine complexes of osmium (II) where the dipyridylamine ligands bears a saturated C(8), C(10), C(12), C(14), or C(16) alkyl chain) were synthesized and characterized electrochemically and biochemically as mediators for glucose oxidase (EC 1.1.3.4, GOD) of Aspergillus niger. These compounds exhibited critical micelle concentrations (CMCs) in phosphate-buffered saline solution (pH 7.4) in the range 10(-4) 10 10(-3) M, the value decreasing with increasing chain length. Dependence of a number of properties (speed of mediation, redox potential, denaturing action on the enzyme, adsorption on an electrode surface) on the length of the mediator alkyl chain was observed. The presence of an alkyl chain decreased the rate of mediation relative to otherwise similar nonsurfactant mediators, and the longer alkyl chain, the slower the rate of mediation. For each compound, mediation above the CMC was about tenfold slower than that observed below the CMC. However, for the cases of mediator absorbed on an electrode surface with GOD, longer chains give increased physisorption of mixed micelles of enzyme and mediator. The compounds were incidentally found to inhibit the glucose oxidase activity of GOD in a complex manner; inhibition increased with increasing chain length and the deactivation, for any given compound, was more pronounced below the CMC than above. Glucose oxidase activity assays and study of the action of surfactants and mediators on the fluorescent properties of carboxy-fluorescein-labeled GOD led to the consideration of a model for redox surfactant-GOD interaction where three mechanisms may operate: first, a selective interaction of mediators with the GOD active site; second, a nondenaturing association of short-chain (相似文献   

Selenium spares ascorbate and alpha-tocopherol in cultured liver cell lines under oxidant stress.

The selenoenzyme thioredoxin reductase (TR) can recycle ascorbic acid, which in turn can recycle alpha-tocopherol. Therefore, we evaluated the role of selenium in ascorbic acid recycling and in protection against oxidant-induced loss of alpha-tocopherol in cultured liver cells. Treatment of HepG2 or H4IIE cultured liver cells for 48 h with sodium selenite (0-116 nmol/l) tripled the activity of the selenoenzyme TR, measured as aurothioglucose-sensitive dehydroascorbic acid (DHA) reduction. However, selenium did not increase the ability of H4IIE cells to take up and reduce 2 mM DHA, despite a 25% increase in ascorbate-dependent ferricyanide reduction (which reflects cellular ascorbate recycling). Nonetheless, selenium supplements both spared ascorbate in overnight cultures of H4IIE cells, and prevented loss of cellular alpha-tocopherol in response to an oxidant stress induced by either ferricyanide or diazobenzene sulfonate. Whereas TR contributes little to ascorbate recycling in H4IIE cells, selenium spares ascorbate in culture and alpha-tocopherol in response to an oxidant stress.     

Probing the turnover efficiency of photosystem II membrane fragments with different electron acceptors

In this study we employ isotope ratio membrane-inlet mass spectrometry to probe the turnover efficiency of photosystem II (PSII) membrane fragments isolated from spinach at flash frequencies between 1Hz and 50Hz in the presence of the commonly used exogenous electron acceptors potassium ferricyanide(III) (FeCy), 2,5-dichloro-p-benzoquinone (DCBQ), and 2-phenyl-p-benzoquinone (PPBQ). The data obtained clearly indicate that among the tested acceptors PPBQ is the best at high flash frequencies. If present at high enough concentration, the PSII turnover efficiency is unaffected by flash frequency of up to 30Hz, and at 40Hz and 50Hz only a slight decrease by about 5-7% is observed. In contrast, drastic reductions of the O(2) yields by about 40% and 65% were found at 50Hz for DCBQ and FeCy, respectively. Comparison with literature data reveals that PPBQ accepts electrons from Q(A)(-) in PSII membrane fragments with similar efficiency as plastoquinone in intact cells. Our data also confirm that at high flashing rates O(2) evolution is limited by the reactions on the electron-acceptor side of PSII. The relevance of these data to the evolutionary development of the water-splitting complex in PSII and with regard to the potential of artificial water-splitting catalysts is discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

A high-potential redox component located within cyanobacterial photosystem II.

The calcium-dependent oxygen evolution activity of preparations of Phormidium luridum shows a marked selectivity in favor of ferricyanide over benzoquinone as Hill oxidant. In addition, the rate of oxygen evolution increases with increasing solution redox potential over the range +350 to +550 mV vs. the standard hydrogen electrode. These properties pertain to both 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive and -insensitive fractions of the total oxygen evolution activity. Neither changes in solution potential nor use of oxidants other than ferricyanide obviate the need for added Ca(2+). To explain these observations, two models are proposed, each of which invokes the existence of a redox component located within Photosystem II and having a midpoint potential greater than +450 mV. In one model, the postulated species is a donor which competes with water for oxidizing equivalents generated by System II. In the other model, the 450 mV species is a high-potential primary acceptor of System II electrons.     

Transient kinetics of electron transfer reactions of flavodoxin: ionic strength dependence of semiquinone oxidation by cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid and computer modeling of reaction complexes

Electron transfer reactions between Clostridum pasteurianum flavodoxin semiquinone and various oxidants [horse heart cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic [horse heart cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid (EDTA)] have been studied as a function of ionic strength by using stopped-flow spectrophotometry. The cytochrome c reaction is complicated by the existence of two cytochrome species which react at different rates and whose relative concentrations are ionic strength dependent. Only the faster of these two reactions is considered here. At low ionic strength, complex formation between cytochrome c and flavodoxin is indicated by a leveling off of the pseudo-first-order rate constant at high cytochrome c concentration. This is not observed for either ferricyanide or ferric EDTA. For cytochrome c, the rate and association constants for complex formation were found to increase with decreasing ionic strength, consistent with negative charges on flavodoxin interacting with the positively charged cytochrome electron transfer site. Both ferricyanide and ferric EDTA are negatively charged oxidants, and the rate data respond to ionic strength changes as would be predicted for reactants of the same charge sign. These results demonstrate that electrostatic interactions involving negatively charged groups are important in orienting flavodoxin with respect to oxidants during electron transfer. We have also carried out computer modeling studies of putative complexes of flavodoxin with cytochrome c and ferricyanide, which relate their structural properties to both the observed kinetic behavior and some more general features of physiological electron transfer processes. The results of this study are consistent with the ionic strength behavior described above.     

Ascorbic acid spares alpha-tocopherol and decreases lipid peroxidation in neuronal cells

Ascorbic acid is considered an antioxidant in the central nervous system, but direct evidence that ascorbate protects neuronal cells from oxidant stress is lacking. Differentiated SH-SY5Y cells in culture took up ascorbic acid on the sodium-dependent vitamin C transporter Type 2 and retained it much more effectively than dehydroascorbic acid. Intracellular ascorbate spared alpha-tocopherol, both in cells loaded with alpha-tocopherol in culture and in cells under oxidant stress due to extracellular ferricyanide. Sparing of alpha-tocopherol in response to ferricyanide was associated with protection against lipid peroxidation in cell membranes. These results show that neuronal cells concentrate ascorbate, and that intracellular ascorbate, either directly or through sparing of alpha-tocopherol, protects them against oxidant stress.     

Redox modulation of reductase and phosphatase activities in human erythrocytes

Summary We have previously reported that ferricyanide reductase activity in human erythrocytes depended on glycolysis and could be modulated by several compounds including oxidants and hormones like insulin. Insulin could activate glycolysis, probably as a consequence of tyrosine phosphorylation of protein band 3, implicating phosphorylation reactions as an important signal for activation of the reductase by insulin. Reversible phosphorylation of cellular proteins is also believed to play a key role in the action of insulin. Cytosolic acid phosphatase activity has been found in human erythrocytes. To further extend initial reports, we studied the effect of modulators on the cytosolic erythrocyte acid phosphatase. Mild oxidants like ferricyanide (1 mM), vanadate (1 mM), Mn²⁺ (0.5 and 1 mM), and phenylarsine oxide (10 and 100 M) inhibited the phosphatase activity. Similarly, insulin at concentrations that stimulate ferricyanide reduction (500, 1000 IU/ml) inhibited the activity of the phosphatase enzyme. The overall results indicated that oxidants are able to inhibit the acid phosphatase and stimulate the redox enzyme. In addition, a significant negative correlation (r = –0.400; P = 0.006) was observed between phosphatase and reductase activities. The observations discussed here, together with previous ones, emphasize that a close association between reductase and phosphatase enzymes may exist and also suggest a role for redox reactions in tyrosine phosphorylation/dephosphorylation-mediated signal transduction pathways.     

A high-potential redox component located within cyanobacterial Photosystem II

The calcium-dependent oxygen evolution activity of preparations of Phormidium luridum shows a marked selectivity in favor of ferricyanide over benzoquinone as Hill oxidant. In addition, the rate of oxygen evolution increases with increasing solution redox potential over the range +350 to +550 mV vs. the standard hydrogen electrode. These properties pertain to both 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive and -insensitive fractions of the total oxygen evolution activity. Neither changes in solution potential nor use of oxidants other than ferricyanide obviate the need for added Ca²⁺.

To explain these observations, two models are proposed, each of which invokes the existence of a redox component located within Photosystem II and having a midpoint potential greater than +450 mV. In one model, the postulated species is a donor which competes with water for oxidizing equivalents generated by System II. In the other model, the 450 mV species is a high-potential primary acceptor of System II electrons.     

Effects of Optically Active 1-(α-Methylbenzyl)-3-(3,4-dichlorophenyl)urea on Reactions of Mitochondria and Chloroplasts

Effects of the R- and S-isomers and racemate of 1-(alpha-methylbenzyl)-3-(3,4-dichlorophenyl)urea (MBPU) were measured on phosphorylation and electron transport in mung bean (Phaseolus aureus L.) mitochondria and spinach (Spinacia oleracea L.) chloroplasts.In chloroplasts, S-MBPU inhibited basal and methylamine-uncoupled electron transport with ferricyanide as the oxidant, both photoreduction and coupled photophosphorylation with water as the electron donor and with ferricyanide and nicotinamide adenine dinucleotide phosphate (NADP) as oxidants, and cyclic photophosphorylation with phenazine methosulfate as the electron mediator under an argon gas phase. With ascorbate 2,6-dichloro-phenolindophenol as the electron donor, phosphorylation coupled to NADP reduction was inhibited, but the reduction of NADP was not inhibited. The R-isomer of MBPU, like the S-isomer, inhibited all of the photophosphorylation reactions studied. However, unlike the S-isomer, the R-isomer either did not inhibit or was a very weak inhibitor of all photoreduction reactions. The effects of the MBPUs on the chloroplast reactions can be explained by action at two different sites: an optically specific site near photosystem II and the oxygen evolution pathway, and a second optically nonspecific site associated with the generation of ATP.In mitochondria, both the R- and S-isomers stimulated state 4 respiration, inhibited state 3 respiration, and released oligomycin-inhibited respiration with malate, succinate, and NADH as substrates. Both enantiomers were equally active in all studies with malate and succinate as substrates. However, with NADH as substrate, R-MBPU was a stronger inhibitor of state 3 respiration and a weaker stimulator of state 4 respiration than S-MBPU.     

Iron stress-induced redox reactions in bean roots

Iron stress-induced and constitutive redox activity of bean ( Phaseolus vulgaris L. cv. Delinel) roots was measured on intact plants using FeEDTA and ferricyanide as electron acceptors. The presence of the translation inhibitor cycloheximide caused a decrease in the reduction of both oxidants. However, a differential decline in the reduction rates of FeEDTA and ferricyanide was observed, suggesting enzyme heterogeneity. In the presence of the H⁺ -ATPase inhibitor vanadate, the reduction of FeEDTA was nearly completely suppressed in both Fe-deficient (–Fe) and Fe-sufficient (+Fe) plants, providing evidence for an involvement of plasma membrane-bound ATPase activity in the regulation of the reduction process. The inhibition of the ferricyanide reduction by vanadate was restricted to –Fe plants.
The data are interpreted in terms of simultaneous operation of distinct redox systems in roots of iron-deficient bean plants. The role of proton extrusion in iron stress-induced electron transfer is discussed.     

Immobilized redox mediators for electrochemical NAD(P)<Superscript>+</Superscript> regeneration

The applicability of dissolved redox mediators for NAD(P)⁺ regeneration has been demonstrated several times. Nevertheless, the use of mediators in solutions for sensor applications is not a very convenient strategy since the analysis is not reagentless and long stabilization times occur. The most important drawbacks of dissolved mediators in biocatalytic applications are interferences during product purification, limited reusability of the mediators, and their cost-intensive elimination from wastewater. Therefore, the use of immobilized mediators has both economic and ecological advantages. This work critically reviews the current state-of-art of immobilized redox mediators for electrochemical NAD(P)⁺ regeneration. Various surface modification techniques, such as adsorption polymerization and covalent linkage, as well as the corresponding NAD(P)⁺ regeneration rates and the operational stability of the immobilized mediator films, will be discussed. By comparison with other existing regeneration systems, the technical potential and future perspectives of biocatalytic redox reactions based on electrochemically fed immobilized mediators will be assessed.     

EM-field effect upon properties of NADPH-cytochrome P-450 reductase with model substrates.

Artificial substrates, including ferricyanide and dichlorophenol indophenol (IP), are frequently used to model the activity of NADPH-cytochrome P-450 reductase, in the xenobiotic-metabolic pathway catalyzed by the P-450 complex. Here, the two oxidants were compared in a microsomal preparation from chicken liver. Low-energy 9.14 GHz perturbation affected both reactions similarly, though the IP reaction may be more sensitive to extremely low energy levels. The reactions of the two oxidants differed from each other in their response to the prior incubation of the microsomes with carbon monoxide and to the presence of superoxide dismutase. The mechanics of the reduction of ferricyanide and the reduction of IP are not identical and the electron-flow paths may be dissimilar. Microwave effect cannot be attributed a temperature change in the reaction medium; it appears to occur at the level of the electron-flow path across the dual-flavin reductase.     

Succinate-ubiquinone reductase linked recycling of alpha-tocopherol in reconstituted systems and mitochondria: requirement for reduced ubiquinone.

Studies have demonstrated that accumulation of mitochondrial tocopheroxyl radical, the primary oxidation product of alpha-tocopherol, accompanies rapid consumption of tocopherol. Enzyme-linked electron flow lowers both the steady-state concentration of the radical and the consumption of tocopherol. Reduction of tocopheroxyl radical by a mitochondrial electron carrier(s) seems a likely mechanism of tocopherol recycling. Succinate-ubiquinone reductase (complex II) was incorporated into liposomes in the presence of tocopherol and ubiquinone-10. After inducing formation of tocopheroxyl radical, it was possible to show that reduced ubiquinone prevents radical accumulation and tocopherol consumption. There was no evidence of direct reduction of tocopheroxyl radical by succinate-reduced complex II. These reactions were also measured using ubiquinone-1 and alpha-C-6-chromanol (2,5,7,8-tetramethyl-2-(4'-methylpentyl)-6-chromanol) which are less hydrophobic analogues of ubiquinone-10 and alpha-tocopherol. Mitochondrial membranes were made deficient in ubiquinone but sufficient in alpha-tocopherol and were reconstituted with added quinone. With these membranes it was shown that mitochondrial enzyme-linked reduction of ubiquinone protects alpha-tocopherol from consumption, and there is a requirement for ubiquinone. This complements the observations made in liposomes and we propose that reduced mitochondrial ubiquinones have a role in alpha-tocopherol protection, presumably through efficient reduction of the tocopheroxyl radical.     

Redox properties of the lipocalin alpha1-microglobulin: reduction of cytochrome c, hemoglobin, and free iron

alpha1-Microglobulin (alpha1m) is a 26-kDa plasma and tissue glycoprotein. The protein has a heterogeneous yellow-brown chromophore consisting of small unidentified prosthetic groups localized to a free thiol group (C34) and three lysyl residues (K92, K118, and K130) around the entrance to a hydrophobic pocket. It was recently reported that alpha1m can bind heme and that a C-terminally processed form of alpha1m degrades heme. It is shown here that alpha1m has catalytic reductase and NADH-dehydrogenase-like activities. Cytochrome c, nitroblue tetrazolium (NBT), methemoglobin, and ferricyanide were reduced by alpha1m. Comparison of the reduction rates suggests that methemoglobin is a better substrate than cytochrome c, NBT, and ferricyanide. The reactions with cytochrome c and NBT were mediated by superoxide anions since they were inhibited by superoxide dismutase. The addition of the biological electron donors NADH, NADPH, or ascorbate enhanced the reduction rate of cytochrome c approximately 30-fold. Recombinant alpha1m, which has much less chromophore than plasma and urine alpha1m, was a stronger reductant than the latter alpha1m forms. Site-directed mutagenesis of C34, K92, K118, and K130 and thiol group chemistry showed that the C34 thiol group was involved in the redox reaction but relies upon cooperation with the lysyl residues. The redox properties of alpha1m may provide a physiological protection mechanism against extracellularly exposed heme groups and other oxidants.