Modification of ferritin during iron loading.

Recombinant human ferritin loaded with iron via its own ferroxidase activity did not sediment through a sucrose-density gradient as a function of iron content. Analysis of the recombinant ferritin by native PAGE demonstrated an increase in altered migration pattern of the ferritins with increasing sedimentation, indicating an alteration of the overall charge of ferritin. Additionally, analysis of the ferritin by SDS-PAGE under nonreducing conditions demonstrated that the ferritin had formed large aggregates, which suggests disulfide bonds are involved in the aggregation. The hydroxyl radical was detected by electron spin resonance spectroscopy during iron loading into recombinant ferritin by its own ferroxidase activity. However, recombinant human ferritin loaded with iron in the presence of ceruloplasmin sedimented through a sucrose-density gradient similar to native ferritin. This ferritin was shown to sediment as a function of iron content. The addition of ceruloplasmin to the iron loading assay eliminated the detection of the DMPO-*OH adduct observed during loading using the ferroxidase activity of ferritin. The elimination of the DMPO-*OH adduct was determined to be due to the ability of ceruloplasmin to completely reduce oxygen to water during the oxidation of the ferrous iron. The implications of these data for the present models for iron uptake into ferritin are discussed.

The consequences of hydroxyl radical formation on the stoichiometry and kinetics of ferrous iron oxidation by human apoferritin.

Despite previous detection of hydroxyl radical formation during iron deposition into ferritin, no reports exist in the literature concerning how it might affect ferritin function. In the present study, hydroxyl radical formation during Fe(II) oxidation by apoferritin was found to be contingent on the "ferroxidase" activity (i.e., H subunit composition) exhibited by apoferritin. Hydroxyl radical formation was found to affect both the stoichiometry and kinetics of Fe(II) oxidation by apoferritin. The stoichiometry of Fe(II) oxidation by apoferritin in an unbuffered solution of 50 mM NaCl, pH 7.0, was approximately 3.1 Fe(II)/O(2) at all iron-to-protein ratios tested. The addition of HEPES as an alternate reactant for the hydroxyl radical resulted in a stoichiometry of about 2 Fe(II)/O(2) at all iron-to-protein ratios. HEPES functioned to protect apoferritin from oxidative modification, for its omission from reaction mixtures containing Fe(II) and apoferritin resulted in alterations to the ferritin consistent with oxidative damage. The kinetic parameters for the reaction of recombinant human H apoferritin with Fe(II) in HEPES buffer (100 mM) were: K(m) = 60 microM, k(cat) = 10 s(-1), and k(cat)/K(m) = 1.7 x 10(5) M(-1) x (-1). Collectively, these results contradict the "crystal growth model" for iron deposition into ferritin and, while our data would seem to imply that the ferroxidase activity of ferritin is adequate in facilitating Fe(II) oxidation at all stages of iron deposition into ferritin, it is important to note that these data were obtained in vitro using nonphysiologic conditions. The possibility that these findings may have physiological significance is discussed.

Role of disulfide bonds in the stability of recombinant manganese peroxidase.

Phanerochaete chrysosporium manganese peroxidase (MnP) [isoenzyme H4] was engineered with additional disulfide bonds to provide structural reinforcement to the proximal and distal calcium-binding sites. This rational protein engineering investigated the effects of multiple disulfide bonds on the stabilization of the enzyme heme environment and oxidase activity. Stabilization of the heme environment was monitored by UV-visible spectroscopy based on the electronic state of the alkaline transition species of ferric and ferrous enzyme. The optical spectral data confirm an alkaline transition to hexacoordinate, low-spin heme species for native and wild-type MnP and show that the location of the engineered disulfide bonds in the protein can have significant effects on the electronic state of the enzyme. The addition of a single disulfide bond in the distal region of MnP resulted in an enzyme that maintained a pentacoordinate, high-spin heme at pH 9.0, whereas MnP with multiple engineered disulfide bonds did not exhibit an increase in stability of the pentacoordinate, high-spin state of the enzyme at alkaline pH. The mutant enzymes were assessed for increased stability by incubation at high pH. In comparison to wild-type MnP, enzymes containing engineered disulfide bonds in the distal and proximal regions of the protein retained greater levels of activity when restored to physiological pH. Additionally, when assayed for oxidase activity at pH 9.0, proteins containing engineered disulfide bonds exhibited slower rates of inactivation than wild-type MnP.

Identification of free radicals produced during phacoemulsification.

PURPOSE: To detect, identify, and quantitate free radicals produced during conditions similar to phacoemulsification cataract surgery. SETTING: Research laboratory at the Biotechnology Center, Utah State University, Logan, Utah, USA. METHODS: All experiments were performed using a Series Ten Thousand phacoemulsifier (Alcon Laboratories) modified to make a 10 mL continuous circulation loop (to increase sensitivity). The irrigating solution was passed through a 3 mL chamber in line with the circulation loop, and electron spin resonance spin trapping methods were used to detect, identify, and quantitate free radical production during phacoemulsification. As an additional indication of hydroxyl radical production, the hydroxylation of salicylate and thiocyanate was detected by high-performance liquid chromatography and spectrophotometry, respectively. RESULTS: The hydroxyl radical was formed when phacoemulsification was performed in the presence of solutions containing spin trap in double deionized water or balanced salt solution (BSS). Hydroxyl radical production was linear with respect to phacoemulsification time. Production of the hydroxyl radical was not observed when phacoemulsification was performed with anaerobic solutions, indicating a requirement for oxygen in radical production. The concentration of trapped hydroxyl radical was reduced in the presence of balanced salt solution with bicarbonate, dextrose, and glutathione (BSS Plus). Upon phacoemulsification, both salicylate and thiocyanate underwent hydroxylation when included in the irrigating solution, confirming the generation of the hydroxyl radical. Additional tests discounted the formation of superoxide or hydrogen peroxide during phacoemulsification. CONCLUSIONS: Hydroxyl radical was produced by phacoemulsification in the presence of aerobic solutions. Hydroxyl radical production was dependent on the presence of molecular oxygen and was not generated as a result of the homolytic cleavage of water. The amount of hydroxyl radical detected was directly proportional to phacoemulsification time and was reduced in the presence of BSS Plus. Other reactive oxygen species such as superoxide, hydrogen peroxide, and ozone were not detected during phacoemulsification under these conditions.

Cellobiose dehydrogenase-an extracellular fungal flavocytochrome.

Wood-degrading fungi, including white-rot and soft-rot fungi as well as at least one brown-rot fungus, produce cellobiose dehydrogenase (CDH). CDH has generated recent interest because of its ability to facilitate the formation of free radicals and because it makes a nice model to study intraprotein electron transfer. While the physiological function of CDH is not known, a considerable portion of this review discusses the strength of the data dealing with individual hypotheses. New evidence dealing with proteolysis of CDH in relationship to the interaction of CDH with lignin and manganese peroxidases are discussed. Additionally, recent information dealing with the catalytic mechanism and reactivity of the individual domains of CDH is detailed.

Measurement of lipid peroxidation.

There is currently considerable interest in what is termed "oxidative stress," or the oxidation of biological macromolecules, with emphasis on its involvement in various diseases and toxicities and methods to limit either its occurrence or effects. This unit describes traditional methods to measure the extent or rate of lipid peroxidations, including assays for conjugated dienes, lipid hydroperoxides, the polyunsaturated lipid breakdown product malondialdehyde, and hemolysis, along with discussion of alternative methods.

Glutathione-mediated mineralization of 14C-labeled 2-amino-4,6-dinitrotoluene by manganese-dependent peroxidase H5 from the white-rot fungus Phanerochaete chrysosporium.

Manganese-dependent peroxidase (MnP) H5 from the white-rot fungus Phanerochaete chrysosporium, in the presence of either Mn(II) (10 mM) or GSH (10 mM). was able to mineralize 14C-U-ring-labeled 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT) up to 29% in 12 days. When both Mn(II) and GSH were present, the mineralization extent reached 82%. On the other hand, no significant mineralization was observed in the absence of both Mn(II) and GSH, suggesting the requirement of a mediator [either Mn(II) or GSH] for the degradation of 2-A-4,6-DNT by MnP. Using electron spin resonance (ESR) techniques, it was found that the glutathionyl free radical (GS*) was produced through the oxidation of GSH by MnP in the presence as well as in the absence of Mn(II). GS* was also generated through the direct oxidation of GSH by Mn(III). Our results strongly suggest the involvement of GS* in the GSH-mediated mineralization of 2-A-4,6-DNT by MnP.

Kinetics and reactivity of the flavin and heme cofactors of cellobiose dehydrogenase from Phanerochaete chrysosporium.

The flavin cofactor within cellobiose dehydrogenase (CDH) was found to be responsible for the reduction of all electron acceptors tested. This includes cytochrome c, the reduction of which has been reported to be by the reduced heme of CDH. The heme group was shown to affect the reactivity and activation energy with respect to individual electron acceptors, but the heme group was not involved in the direct transfer of electrons to substrate. A complicated interaction was found to exist between the flavin and heme of cellobiose dehydrogenase. The addition of electron acceptors was shown to increase the rate of flavin reduction and the electron transfer rate between the flavin and heme. All electron acceptors tested appeared to be reduced by the flavin domain. The addition of ferric iron eliminated the flavin radical present in reduced CDH, as detected by low temperature ESR spectroscopy, while it increased the flavin radical ESR signal in the independent flavin domain, more commonly referred to as cellobiose:quinone oxidoreductase (CBQR). Conversely, no radical was detected with either CDH or CBQR upon the addition of methyl-1,4-benzoquinone. Similar reaction rates and activation energies were determined for methyl-1,4-benzoquinone with both CDH and CBQR, whereas the rate of iron reduction by CDH was five times higher than by CBQR, and its activation energy was 38 kJ/mol lower than that of CBQR. Oxygen, which may be reduced by either one or two electrons, was found to behave like a two-electron acceptor. Superoxide production was found only upon the inclusion of iron. Additionally, information is presented indicating that the site of substrate reduction may be in the cleft between the flavin and heme domains.

Intact human ceruloplasmin is required for the incorporation of iron into human ferritin.

We have previously reported several studies on the loading of iron into ferritin by ceruloplasmin using proteins from rats. Loading iron into human ferritin using human serum ceruloplasmin is complicated by the fact that human ceruloplasmin is very susceptible to proteolysis (T. P. Ryan, T. A. Grover, and S. D. Aust, 1992, Arch. Biochem Biophys. 293, 1-8). The present study investigated the effect of proteolysis on the ability of human ceruloplasmin to load iron into human ferritin. SDS-PAGE revealed one major band with an apparent molecular weight of 116 kDa for a proteolytically degraded form of ceruloplasmin versus a 132-kDa band for an intact form of the enzyme. Both forms of the enzyme possessed ferroxidase activity, although that of the proteolytically degraded enzyme was approximately twofold less than that of the intact enzyme (4.9 nmol (min)-1 vs 8.3 nmol (min)-1). Only the intact form of ceruloplasmin was able to catalyze iron loading into ferritin without altering the physical characteristics of the ferritin protein during the process. Abnormal migration in nondenaturing PAGE gels, as well as a decrease in the amount of detectable ferritin protein, was observed when ferritin was incubated with iron alone or with proteolytically degraded ceruloplasmin and iron. It was concluded that the structural integrity of ceruloplasmin is required for the enzyme to effectively catalyze iron loading into ferritin.

Engineering a disulfide bond in recombinant manganese peroxidase results in increased thermostability.

Manganese peroxidase (MnP) produced by Phanerochaete chrysosporium, which catalyzes the oxidation of Mn(2+) to Mn(3+) by hydrogen peroxide, was shown to be susceptible to thermal inactivation due to the loss of calcium [Sutherland, G. R. J.; Aust, S. D. Arch. Biochem. Biophys. 1996, 332, 128-134]. The recombinant enzyme, lacking glycosylation, was found to be more susceptible [Nie, G.; Reading, N. S.; Aust, S. D. Arch. Biochem. Biophys. 1999, 365, 328-334]. On the basis of the properties and structure of peanut peroxidase, we have engineered a disulfide bond near the distal calcium binding site of MnP by means of the double mutation A48C and A63C. The mutant enzyme had activity and spectral properties similar to those of native, glycosylated MnP. The thermostabilities of native, recombinant, and mutant MnP were studied as a function of temperature and pH. MnPA48C/A63C exhibited kinetics of inactivation similar to that of native MnP. The addition of calcium decreased the rate of thermal inactivation of the enzymes, while EGTA increased the rate of inactivation. Thermally treated MnPA48C/A63C mutant was shown to contain one calcium, and it retained a percentage of its original manganese oxidase activity; native and recombinant MnP were inactivated by the removal of calcium from the protein.

Substrate specificity of lignin peroxidase and a S168W variant of manganese peroxidase.

Lignin peroxidase (LiP) and manganese peroxidase (MnP) are structurally similar heme-containing enzymes secreted by white-rot fungi. Unlike MnP, which is only specific for Mn(2+), LiP has broad substrate specificity, but it is not known if this versatility is due to multiple substrate-binding sites. We report here that a S168W variant of MnP from Phanerochaete chrysosporium not only retained full Mn(2+) oxidase activity, but also, unlike native or recombinant MnP, oxidized a multitude of LiP substrates, including small molecule and polymeric substrates. The kinetics of oxidation of most nonpolymeric substrates by the MnP variant and LiP were similar. The stoichiometries for veratryl alcohol oxidation by these two enzymes were identical. Some readily oxidizable substrates, such as guaiacol and ferrocyanide, were oxidized by MnP S168W and LiP both specifically and nonspecifically while recombinant MnP oxidized these substrates only nonspecifically. The functional similarities between this MnP variant and LiP provide evidence for the broad substrate specificity of a single oxidation site near the surface tryptophan.

Enzymology of Phanerochaete chrysosporium with respect to the degradation of recalcitrant compounds and xenobiotics.

The archetypal white-rot fungus Phanerochaete chrysosporium has been shown to degrade a variety of persistent environmental pollutants. Many of the enzymes responsible for pollutant degradation, which are normally involved in the degradation of wood, are extracellular. Thus, P. chrysosporium is able to degrade toxic or insoluble chemicals more efficiently than other microorganisms. P. chrysosporium has a range of oxidative and reductive mechanisms and uses highly reactive, nonspecific redox mediators which increase the number of chemicals that can be effectively degraded. This review gives an overview of the enzymes that are believed to be important for bioremediation and briefly discusses the degradation of some individual chemicals.

Production of recombinant human apoferritin heteromers.

We are interested in learning how iron is safely inserted and stored in ferritin. Recombinant DNA technology has considerable potential in determining the functional roles of the two ferritin subunits (H and L). In previous studies, we have observed that recombinant rat H ferritin was repressive to cell growth in both prokaryotic and eukaryotic expression systems (Guo et al., Biochem. Biophys. Res. Commun. 242, 39-45 (1998)). This results in the protein being expressed at very low levels. This problem was partially bypassed by the use of an inducible expression system, which utilizes T7 RNA polymerase dependent expression of the gene, induced by isopropyl beta-D-thiogalactopyranoside (IPTG). Simultaneously expressing the H and L ferritin genes in this system resulted in only a narrow range of ferritin heteromers, which predominantly consisted of the L subunit. Addition of rifampicin to cultures, 1 h following the induction of protein synthesis by IPTG, increased the production of the H subunit and thus increased the range of ferritin H:L subunit ratios. Simultaneous expression of the H and L ferritin genes in Escherichia coli grown in a deficient medium with minimal iron and with the addition of rifampicin resulted in the production of a range of recombinant human apoferritin heteromers that could be separated based on their subunit composition.

Biodegradation of superabsorbent polymers in soil.

Biodegradation of two superabsorbent polymers, a crosslinked, insoluble polyacrylate and an insoluble polyacrylate/ polyacrylamide copolymer, in soil by the white-rot fungus, Phanerochaete chrysosporium was investigated. The polymers were both solubilized and mineralized by the fungus but solubilization and mineralization of the copolymer was much more rapid than of the polyacrylate. Soil microbes poorly solublized the polymers and were unable to mineralize either intact polymer. However, soil microbes cooperated with the fungus during polymer degradation in soil, with the fungus solubilizing the polymers and the soil microbes stimulating mineralization. Further, soil microbes were able to significantly mineralize both polymers after solubilization by P. chrysosporium grown under conditions that produced fungal peroxidases or cellobiose dehydrogenase, or after solubilization by photochemically generated Fenton reagent. The results suggest that biodegradation of these polymers in soil is best under conditions that maximize solubilization.

Cellobiose dehydrogenase-dependent biodegradation of polyacrylate polymers by Phanerochaete chrysosporium.

When Phanerochaete chrysosporium was cultured using conditions which promote the expression of cellobiose dehydrogenase (CDH), but not the ligninolytic peroxidases, the fungus effectively solubilized and mineralized an insoluble, crosslinked polyacrylate and an insoluble polyacrylate/polyacrylamide copolymer. Addition of iron to the cultures increased CDH activity in the cultures and the rate and extent of solubilization and mineralization of both polymers. Solubilization of both polymers was observed when incubated with purified CDH, ferric iron and hydrogen peroxide.

Degradation of chemicals by reactive radicals produced by cellobiose dehydrogenase from Phanerochaete chrysosporium.

Phanerochaete chrysosporium, grown on cellulose, produced a cellobiose-dependent dehydrogenase which reduced both ferric iron and molecular oxygen, resulting in the generation of the hydroxyl radical. The hydroxyl radical was detected in reaction mixtures with and without the addition of exogenous H2O2. The purified reductase and the fungus grown under nonligninolytic conditions that promote the production of the reductase were able to depolymerize an insoluble polyacrylate polymer. When oxalate, a secondary metabolite of P. chrysosporium, was used as the iron chelator, it was oxidized by the hydroxyl radical to form the carboxylate anion radical, a strong reductant. Under these reductive conditions, the enzyme was shown to catalyze the reduction of bromotrichloromethane to the trichloromethyl radical. We propose that these oxidative and reductive mechanisms may contribute to the degradation of a wide range of environmental pollutants by fungi which produce this enzyme.

Relative stability of recombinant versus native peroxidases from Phanerochaete chrysosporium.

Two types of glycosylated peroxidases are secreted by the white-rot fungus Phanerochaete chrysosporium, lignin peroxidase (LiP) and manganese peroxidase (MnP). The thermal stabilities of recombinant LiPH2, LiPH8, and MnPH4, which were expressed without glycosylation in Escherichia coli, were lower than those of corresponding native peroxidases isolated from P. chrysosporium. Recovery of thermally inactivated recombinant enzyme activities was higher than with that of the thermally inactivated native peroxidases. Removal of N-linked glycans from native LiPH8 and MnPH4 did not affect enzyme activities or thermal stabilities of the enzymes. Although LiPH2, LiPH8, and MnPH4 contained O-linked glycans, only the O-linked glycans from MnPH4 could be removed by O-glycosidase, and the glycan-depleted MnPH4 exhibited essentially the same activity as nondeglycosylated MnPH4, but thermal stability decreased. Periodate-treated MnPH4 exhibited even lower thermal stability than O-glycosidase treated MnPH4. The role of O-linked glycans in protein stability was also evidenced with LiPH2 and LiPH8. Based on these data, we propose that neither N- nor O-linked glycans are likely to have a direct role in enzyme activity of native LiPH2, LiPH8, and MnPH4 and that only O-linked glycans may play a crucial role in protein stability of native peroxidases.

Addition of veratryl alcohol oxidase activity to manganese peroxidase by site-directed mutagenesis.

Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungus Phanerochaete chrysosporium. Despite structural similarity, these peroxidases oxidize different substrates. Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+. By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed in Escherichia coli. The kcat for veratryl alcohol oxidation was 11 s-1, Km for veratryl alcohol approximately 0.49 mM, and Km for hydrogen peroxide approximately 25 microM at pH 2.3. The Km for veratryl alcohol was higher and Km for hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes. The mutant retained full manganese peroxidase activity and the kcat was approximately 2.6 x 10(2) s-1 at pH 4.3. Consistent with relative activities with respect to these substrates, Mn2+ strongly inhibited veratryl alcohol oxidation. The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis.

Mutational analysis of loading of iron into rat liver ferritin by ceruloplasmin.

Site-directed mutagenesis was used to investigate the loading of iron into rat liver ferritin by ceruloplasmin. Changes were made in the H chain to investigate the role of tyrosines involved in an inherent ferroxidase activity thought to be involved in the self-loading of iron into ferritin. Mutation Y34F affected the rate of iron loading by ceruloplasmin and incorporation of the oxidized iron into the core. Mutation Y29R (making it analogous to the L chain) had no effect on iron oxidation but slightly decreased core formation. A double mutation in the L chain, to open the alpha-helix bundle channel, and R25Y, making the protein more analogous to the H chain, increased the amount of iron incorporated into the core, again suggesting that this Tyr is involved in ligand exchange for core formation. Additional changes in the L chain involving the BC loop suggest that the entire BC loop is involved in the association of ferritin with ceruloplasmin, increasing its ferroxidase activity and the rate of iron loading into ferritin.