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PubMed1975

[Comparative studies on the action of chlorine and ozone on polioviruses in the reprocessing of drinking water in Essen (author's transl)].

The present study was performed in order to evaluate comparatively the inactivation of polio virus type I strains (wild type and attenuated) by means of chlorine and ozone. Polio virus type I was chosen with regard to its epidemiological behaviour and high stability in drinking-water and sewage lines. In view of the lack of propagation techniques, hepatitis viruses A and B, unfortunately, could not be used for these experiments. The experiments were done under laboratory conditions only, and not in the water recovery plant because of hygienic reasons. Defined quantities of disinfectants were examined for their virus-inactivating effect in water without redox-potential (double-distilled water), water with low defined redox-potential (double-distilled water + KOH), previously chlorinated water with a residual chlorine content of 0.03 mg chlorine per liter (tap water) and water with a high redox-potential (well water from the drinking-water plant). Time-course studies were performed, both with chlorine and ozone, in order to evaluate the characteristics of the inactivation procedure. The experimental conditions chosen varied from experiment to experiment to obtain relevant conclusions for the practice. On the basis of our results, and taking into account the quantitative differences in effect, chlorine and ozone principially can be considered equivalent in their action of virus-disinfection. Both, the initial rate and the kinetics of virus disinfection are really identical. Both disinfectants are dependant on the condition of the water (redox-potential, pH etc.) to a great extent in their efficacy. Therefore, a decision of whether or not ozone should substitute for chlorine for the drinking-water supply in Essen cannot be drawn on the basis of virological experiments. This decision, then, depends more or less on other questions - such as relative costs and practicability of the ozonization on a large technical scale. The safety risk and technical reliability of the ozonization process is of particular significance. In the present condition of the Essen reservoir water, a good virus disinfection can be expected already with 1.0 to 1.5 mg ozone/liter (dissolved!); such a concentration guarantees very little residual ozone and, thus, makes then this procedure technically feasible. Continuous checking of the redox-potential and the amount of the ozone added is necessary. With regard to a continuous supply of ozone, the dependence on current supply must be guaranteed. Ozonization of water, probably by the cleavage of humic acid, promotes bacterial recontamination of the drinking-water in the city taps(Stalder und Klosterkötter, 45). Therefore only a combined pre-ozonization with subsequent chlorination would guarantee, both, safety and improvement of the cosmetical conditions of the drinking-water. Such a combination would be feasible with highly reduced amounts of ozone and chlorine.

Chlorine↗
PubMed2006

The role of a conserved serine residue within hydrogen bonding distance of FAD in redox properties and the modulation of catalysis by Ca2+/calmodulin of constitutive nitric-oxide synthases.

The crystal structure of the neuronal nitric-oxide synthase (nNOS) NADPH/FAD binding domain indicated that Ser-1176 is within hydrogen bonding distance of Asp-1393 and the O4 atom of FAD and is also near the N5 atom of FAD (3.7 A). This serine residue is conserved in most of the ferredoxin-NADP+ reductase family of proteins and is important in electron transfer. In the present study, the homologous serines of both nNOS (Ser-1176) and endothelial nitric-oxide synthase (eNOS) (Ser-942) were mutated to threonine and alanine. Both substitutions yielded proteins that exhibited decreased rates of electron transfer through the flavin domains, in the presence and absence of Ca2+/CaM, as measured by reduction of potassium ferricyanide and cytochrome c. Rapid kinetics measurements of flavin reduction of all the mutants also showed a decrease in the rate of flavin reduction, in the absence and presence of Ca2+/CaM, as compared with the wild type proteins. The serine to alanine substitution caused both nNOS and eNOS to synthesize NO more slowly; however, the threonine mutants gave equal or slightly higher rates of NO production compared with the wild type enzymes. The midpoint redox potential measurements of all the redox centers revealed that wild type and threonine mutants of both nNOS and eNOS are very similar. However, the redox potentials of the FMN/FMNH* couple for alanine substitutions of both nNOS and eNOS are >100 mV higher than those of wild type proteins and are positive. These data presented here suggest that hydrogen bonding of the hydroxyl group of serine or threonine with the isoalloxazine ring of FAD and with the amino acids in its immediate milieu, particularly nNOS Asp-1393, affects the redox potentials of various flavin states, influencing the rate of electron transfer.

Alanine↗
PubMed1991

Analysis of the influence of aeroions on ECG and correlation between this action and tissue redox-state potential.

It was observed in rats, that following negative aeroionization heart frequency and the altitude of P-waves increased. After positive ionization these interrelationships took place inversely. As between these effects and the tendency of tissue redox-state potential changes correlation was seen, the results were grouped also on redox basis, independently on whether the increase or decrease of redox-state potential was caused by negative or positive aeroions. The results of this grouping showed, that following an elevation of tissue redox-state potential (+delta E'0) heart frequency dropped, and the altitude of T-waves increased. After a decrease of tissue redox-state potential (-delta E'0) these interrelationships were realized inversely. After -delta E'0 the positive chronotropic influence of noradrenaline increased, but consequent to +delta E'0 the classical positive chronotropic effect of this catecholamine was reversed. These results corroborate our earlier notion, that aeroions exert their action on heart through changing tissue redox-state potential.

Animals↗
PubMed2006

Synthetic seleno-glutaredoxin 3 analogues are highly reducing oxidoreductases with enhanced catalytic efficiency.

Selenoenzymes have a central role in maintaining cellular redox potential. These enzymes have selenenylsulfide bonds in their active sites that catalyze the reduction of peroxides, sulfoxides, and disulfides. The selenol/disufide exchange reaction is common to all of these enzymes, and the active site redox potential reflects the ratio between the forward and reverse rates of this reaction. The preparation of enzymes containing selenocysteine (Sec) is experimentally challenging. As a result, little is known about the kinetic role of selenols in enzyme active sites, and the redox potential of a selenenylsulfide or diselenide bond in a protein has not been experimentally determined. To fully evaluate the effects of Sec on oxidoreductase redox potential and kinetics, glutaredoxin 3 (Grx3) and all three Sec variants of its conserved (11)CXX(14)C active site were chemically synthesized. Grx3, Grx3(C11U), and Grx3(C14U) exhibited redox potentials of -194, -260, and -275 mV, respectively. The position of redox equilibrium between Grx3(C11U-C14U) (-309 mV) and thioredoxin (Trx) (-270 mV) suggests a possible role for diselenide bonds in biological systems. Kinetic analysis is consistent with the hypothesis that the lower redox potentials of the Sec variants result primarily from the greater nucleophilicity of the active site selenium rather than its role as either a leaving group or a "central atom" in the exchange reaction. The 10(2)-10(4)-fold increase in the rate of Trx reduction by the seleno-Grx3 analogues demonstrates that oxidoreductases containing either selenenyl-sulfide or diselenide bonds can have physiologically compatible redox potentials and enhanced reduction kinetics in comparison with their sulfide counterparts.

Binding Sites↗
PubMed2004

Roles of noncoordinated aromatic residues in redox regulation of cytochrome c3 from Desulfovibrio vulgaris Miyazaki F.

The roles of aromatic residues in redox regulation of cytochrome c(3) were investigated by site-directed mutagenesis at every aromatic residue except for axial ligands (Phe20, Tyr43, Tyr65, Tyr66, His67, and Phe76). The mutations at Phe20 induced large chemical shift changes in the NMR signals for hemes 1 and 3, and large changes in the microscopic redox potentials of hemes 1 and 3. The NMR signals of the axial ligands of hemes 1 and 3 were also affected. Analysis of the nature of the mutations revealed that a hydrophobic environment and aromaticity are important for the reduction of the redox potentials of hemes 1 and 3, respectively. There was also a global effect. The replacement of Tyr66 with leucine induced chemical shift changes for heme 4, and changes in the microscopic redox potentials of heme 4. The mutations of Tyr65 induced changes in the chemical shifts and microscopic redox potentials for every heme, suggesting that Tyr65 stabilizes the global conformation, thereby reducing the redox potentials. In contrast, although the mutations of His67 and Phe76 caused chemical shift changes for heme 2, they did not affect its redox potentials, showing these residues are not important. All noncoordinated aromatic residues conserved in the cytochrome c(3) subfamily with heme binding motifs CXXCH, CXXXXCH, CXXCH, and CXXXXCH (Phe20, Tyr43, and Tyr66) are involved in the pi-pi interaction, which causes a decrease in the redox potential of the interacting heme. The global effect can be attributed to either direct or indirect interactions among the four hemes in the cyclic architecture.

Amino Acid Sequence↗
PubMed2000

On the interpretation of quantitative structure-function activity relationship data for lactate oxidase.

The native flavin, FMN, has been removed from the l-lactate oxidase of Aerococcus viridans, and the apoprotein reconstituted with 12 FMN derivatives with various substituents at the flavin 6- and 8-positions. Impressive linear relationships are exhibited between the sum of the Hammett final sigma(para) and final sigma(ortho) parameters and the redox potentials of the free flavins, and between the redox potentials of the free and enzyme-bound flavins. Rapid reaction kinetics studies of the reconstituted enzymes with the substrates l-lactate and l-mandelate show an increase in the reduction rate constant with increasing redox potential, except that, with lactate, a limiting rate constant of approximately 700 s(-1) is obtained with flavins of high potential. Similar breakpoints are found in plots of the rate constants for flavin N5-sulfite adduct formation and for the reaction of the reduced enzymes with molecular oxygen. These results are interpreted in terms of a two-step equilibrium preceding the chemical reaction step, in which the second equilibrium step provides an upper limit to the rate with which the particular substrate or ligand is positioned with the flavin in the correct fashion for the observed chemical reaction to occur. The relationship of rate constants for flavin reduction and N5-sulfite adduct formation with flavin redox potential below the observed breakpoint indicate development of significant negative charge in the transition states of the reactions. In the case of reduction by substrate, the results are consistent either with a hydride transfer mechanism or with the so called "carbanion" mechanism, in which the substrate alpha-proton is abstracted by an enzyme base protected from exchange with solvent. These conclusions are supported by substrate alpha-deuterium isotope effects and by solvent viscosity effects on sulfite binding.

Apoenzymes↗
PubMed2000

Redox state of glutathione in human plasma.

Thiol and disulfide forms of glutathione (GSH) and cysteine (Cys) were measured in plasma from 24 healthy individuals aged 25-35 and redox potential values (E(h)) for thiol/disulfide couples were calculated using the Nernst equation. Although the concentration of GSH (2.8 +/- 0.9 microM) was much greater than that of GSSG (0.14 +/- 0.04 microM), the redox potential of the GSSG/2GSH pool (-137 +/- 9 mV) was considerably more oxidized than values for tissues and cultured cells (-185 to -258 mV). This indicates that a rapid oxidation of GSH occurs upon release into plasma. The difference in values between individuals was remarkably small, suggesting that the rates of reduction and oxidation in the plasma are closely balanced to maintain this redox potential. The redox potential for the Cys and cystine (CySS) pool (-80 +/- 9 mV) was 57 mV more oxidized, showing that the GSSG/2GSH and the CySS/2Cys pools are not in redox equilibrium in the plasma. Potentials for thiol/disulfide couples involving CysGly were intermediate between the values for these couples. Regression analyses showed that the redox potentials for the different thiol/disulfide couples within individuals were correlated, with the E(h) for CySS-mono-Gly/(Cys. CysGly) providing the best correlation with other low molecular weight pools as well as protein disulfides of GSH, CysGly and Cys. These results suggest that E(h) values for GSSG/2GSH and CySS-mono-Gly/(Cys. CysGly) may provide useful means to quantitatively express the oxidant/antioxidant balance in clinical and epidemiologic studies.

Adult↗
PubMed1996

Redox properties of wild-type, Cys69Ala, and Cys69Ser Azotobacter vinelandii flavodoxin II as measured by cyclic voltammetry and EPR spectroscopy,.

This study deals with the detailed electrochemistry and complete EPR-monitored titrations of flavodoxin II of Azotobacter vinelandii (ATCC 478). Since wild-type flavodoxin dimerises via intermolecular disulphide bond formation between Cys69 residues, Cys69 has been replaced by both an alanine and a serine residue. Redox properties of the C69A and C69S flavodoxin mutants were compared to those of wild-type flavodoxin. In the presence of the promotor neomycin, C69A and C69S flavodoxin showed a reversible response of the semiquinone/hydroquinone couple at the glassy carbon electrode. However, the addition of dithiothreitol proved to be necessary for the stabilisation of the wild-type flavodoxin response. EPR-monitored redox titrations of wild-type and C69A flavodoxin at high and low pH confirmed the redox potentials measured using cyclic voltammetry. The pH dependence of the semiquinone/hydroquinone redox potentials cannot be described using a model assuming one redox-linked pK. Instead, the presence of at least two redox-linked protonation sites is suggested: pKred.1 = 5.39 +/- 0.08, pKox = 7.29 +/- 0.14, and pKred.2 = 7.84 +/- 0.14 with Em.7 = -459 +/- 4 mV, and a constant redox potential at high pH of -485 +/- 4 mV. The dependence of the semiquinone/hydroquinone redox potential on temperature is -0.5 +/- 0.1 mV . K(-1), yielding delta H degrees = 28.6 +/- 1.5 kJ . mol(1) and delta S degrees = -50.0 +/- 6.2 J . mol(-1) . K(-1). No significant differences in redox properties of wild-type, C69A, and C69S flavodoxin were observed. The electrochemical data suggest that replacement of Cys69 in the vicinity of the FMN by either an alanine or a serine residue does not alter the dielectric properties and structure of A. vinelandii flavodoxin II.

Azotobacter vinelandii↗
PubMed1995

Lipoamide dehydrogenase from Escherichia coli lacking the redox active disulfide: C44S and C49S. Redox properties of the FAD and interactions with pyridine nucleotides.

The cysteines that comprise the active site disulfide in lipoamide dehydrogenase have been individually mutated to a serine residue to give the altered enzymes, C44S and C49S, making it possible to study the redox behavior of the FAD in the absence of the disulfide. The redox potential of the FAD in C44S and C49S was -379 and -345 mV, respectively, at pH 7.0, 25 degrees C. A plot of the redox potential as a function of pH for C49S gave slopes of 57 mV/pH from pH 5.0 to 7.9 and 10 mV/pH from pH 7.9 to 8.8. The plot of the redox potential as a function of pH for C44S gave slopes of 70 mV/pH from pH 5.0 to 7.9 and 4 mV/pH from pH 7.9 to 8.38. The change in the slope at pH 7.9 is associated with the ionization (pKa) of the FADH2 to FADH- in the reduced form of both enzymes. These determinations show that the redox potential of the FAD in C49S, in C44S, and in wild type enzyme is modulated by the electronegativity of its nearest neighbor, hydroxyl, thiolate, or disulfide, and that the flavin is bound more tightly to the oxidized forms of these enzymes than to the reduced forms. The redox potentials of these enzymes determined using NADH and NADPH at pH 7.6, 25 degrees C are as follows: C44S, -350 mV, -369 mV; C49S, -328 mV, -353 mV, respectively. Thus, pyridine nucleotide binding raises the redox potential of the flavin, showing that both substrates bind more tightly to the reduced form of the enzymes, as well as tighter binding of NADH to the enzymes than that of NADPH. Kd values for the binding of NADH and NADPH to oxidized C44S and C49S were determined in pre-steady-state kinetics at pH 7.6 and 25 degrees C, which were monophasic when NADPH was the reductant and biphasic with NADH. The binding constants for NADPH were 660 microM for C44S and 500 microM for C49S; using NADH, the binding constants were 137 microM for C44S and 23 microM for C49S. Fluorescence and absorbance spectrophotometry were used to determine the binding of NAD+ to the oxidized forms of the enzymes as 275 microM and 270 microM for C44S and C49S, respectively.

Binding Sites↗
PubMed2004

Oxidation-reduction potential regulates RpoS levels in Salmonella Typhimurium.

AIMS: The aim of this work was to investigate the connection between oxidation-reduction (redox) potential and stationary phase induction of RpoS in Salmonella Typhimurium. METHODS AND RESULTS: A lux-based reporter was used to evaluate RpoS activity in S. Typhimurium pure cultures. During growth of S. Typhimurium, a drop in the redox potential of the growth medium occurred at the same time as RpoS induction and entry into stationary phase. An artificially induced decrease in redox potential earlier during growth reduced the time to RpoS induction and Salmonella entered the stationary phase prematurely. In contrast, under high redox conditions, Salmonella grew unaffected and entered the stationary growth phase as normal, although RpoS induction did not occur. As a consequence, stationary phase cells grown in the high redox environment were significantly more heat sensitive (P < 0.05) than those grown under normal conditions. CONCLUSIONS: This work suggests that redox potential can regulate RpoS levels in S. Typhimurium and can thus, control the expression of genes responsible for thermal resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to manipulate RpoS induction and control stationary phase gene expression can have important implications in food safety. Early RpoS induction under low redox potential conditions can lead to enhanced resistance in low cell concentrations to inimical processes such as heat stress. Inhibition of RpoS induction would abolish stationary phase protective properties making cells more sensitive to common food control measures.

Bacterial Proteins↗
PubMed2005

Enhancement of quinone redox cycling by ascorbate induces a caspase-3 independent cell death in human leukaemia cells. An in vitro comparative study.

Since the higher redox potential of quinone molecules has been correlated with enhanced cellular deleterious effects, we studied the ability of the association of ascorbate with several quinones derivatives (having different redox potentials) to cause cell death in K562 human leukaemia cell line. The rationale is that the reduction of quinone by ascorbate should be dependent of the quinone half-redox potential thus determining if reactive oxygen species (ROS) are formed or not, leading ultimately to cell death or cell survival. Among different ROS that may be formed during redox cycling between ascorbate and the quinone, the use of different antioxidant compounds (mannitol, desferal, N-acetylcysteine, catalase and superoxide dismutase) led to support H2O2 as the main oxidizing agent. We observed that standard redox potentials, oxygen uptake, free ascorbyl radical formation and cell survival were linked. The oxidative stress induced by the mixture of ascorbate and the different quinones decreases cellular contents of ATP and GSH while caspase-3-like activity remains unchanged. Again, we observed that quinones having higher values of half-redox potential provoke a severe depletion of ATP and GSH when they were associated with ascorbate. Such a drop in ATP content may explain the lack of activation of caspase-3. In conclusion, our results indicate that the cytotoxicity of the association quinone/ascorbate on K562 cancer cells may be predicted on the basis of half-redox potentials of quinones.

Adenosine Triphosphate↗
PubMed1999

Role of the axial ligand in type 1 Cu centers studied by point mutations of met148 in rusticyanin.

Type 1 Cu centers in cupredoxins, nitrite reductases, and multi-copper oxidases utilize the same trigonal core ligation to His-Cys-His, with a weak axial ligand generally provided by a Met sulfur. In azurin, an additional axial ligand, a carbonyl oxygen from a Gly, is present. The importance of these axial ligands and in particular the Met has been debated extensively in terms of their role in fine-tuning the redox potential, spectroscopic properties, and rack-induced or entatic state properties of the copper sites. Extensive site-directed mutagenesis of the Met ligand has been carried out in azurin, but the presence of an additional carbonyl oxygen axial ligand has made it difficult to interpret the effects of these substitutions. Here, the axial methionine ligand (Met148) in rusticyanin is replaced with Leu, Gln, Lys, and Glu to examine the effect on the redox potential, acid stability, and copper site geometry. The midpoint redox potential varies from 363 (Met148Lys) to 798 mV (Met148Leu). The acid stability of the oxidized proteins is reduced except for the Met148Gln mutant. The Gln mutant remains blue at all pH values between 2.8 and 8, and has a redox potential of 563 mV at pH 3.2. The optical and rhombic EPR properties of this mutant closely resemble those of stellacyanin, which has the lowest redox potential among single-type 1 copper proteins (185 mV). The Met148Lys mutant exhibits type 2 Cu EPR and optical spectra in this pH range. The Met148Glu mutant exhibits a type 2 Cu EPR spectrum above pH 3 and a mixture of type 1 and type 2 Cu spectra at lower pH. The Met148Leu mutant exhibits the highest redox potential ( approximately 800 mV at pH 3.2) which is similar to the values in fungal laccase and in the type 1 Cu site of ceruloplasmin where this axial ligand is also a Leu.

Azurin↗
PubMedDate not supplied

[Physico-chemical properties of aqueous solutions, prepared in a membrane electrolyzer].

The physicochemical properties of aqueous solutions resulting from membrane electrolysis were studied. It was shown that the catholyte contains hydrogen peroxide at a concentration of 10(-7), which is formed during the reduction of soluble oxygen. It was found that the relaxation of the catholyte redox potential is caused by the transition of the reducing agent to the gaseous phase. The relaxation characteristics of the redox potentials of the catholyte and molecular hydrogen solution were compared. The similarity of the relaxation characteristics of the catholyte and the hydrogen solution as well as the fact that the catholyte, despite its low redox potential, does not reduce either potassium ferricyanide or 5-5'-dithiobis(2-nitrobenzoic acid) support the suggestion that the redox potential of the catholyte is due to molecular hydrogen. However, based on this suggestion, it is impossible to explain the increase in the relaxation time of the catholyte with increasing ionic strength and the fact that, as the redox potential of the catholyte decreases, the concentration of other gases dissolved in the catholyte remains unchanged. Thus, the question regarding the nature of the reducing agent remains open.

Dithionitrobenzoic Acid↗
PubMed2000

Use of stopped-flow spectrophotometry to establish midpoint potentials for redox proteins.

Stopped-flow spectrophotometry was examined as a tool to assign midpoint potentials to protein redox half-reactions. The method involves the rapid mixing of protein and electron transfer mediator solutions and the determination of the absorbance of at least one of the reacting species or products at equilibrium. The utility of the method was demonstrated with two different redox proteins (nitrogenase iron protein and cytochrome c) with very different midpoint potentials. The overall errors ranged from about +/-0.5 to 3 mV. Advantages of the method include short times required for the experiments, the precision and accuracy of the method in comparison to other methods, the conservative use of valuable protein in the experiments and the ease of obtaining midpoint potentials for redox protein half-reactions, and the potential range covered by a single electron transfer mediator when the method involves mediated electron transfer. It is concluded that the stopped-flow spectrophotometry should be considered the method of choice for determining protein midpoint potentials.

Adenosine Diphosphate↗
PubMed2006

Atomic resolution crystal structures, EXAFS, and quantum chemical studies of rusticyanin and its two mutants provide insight into its unusual properties.

Rusticyanin from the extremophile Thiobacillus ferrooxidans is a blue copper protein with unusually high redox potential and acid stability. We present the crystal structures of native rusticyanin and of its Cu site mutant His143Met at 1.27 and 1.10 A, respectively. The very high resolution of these structures allows a direct comparison with EXAFS data and with quantum chemical models of the oxidized and reduced forms of the proteins, based upon both isolated and embedded clusters and density functional theory (DFT) methods. We further predict the structure of the Cu(II) form of the His143Met mutant which has been experimentally inaccessible due to its very high redox potential. We also present metrical EXAFS data and quantum chemical calculations for the oxidized and reduced states of the Met148Gln mutant, this protein having the lowest redox potential of all currently characterized mutants of rusticyanin. These data offer new insights into the structural factors which affect the redox potential in this important class of proteins. Calculations successfully predict the structure and the order of redox potentials for the three proteins. The calculated redox potential of H143M ( approximately 400 mV greater than native rusticyanin) is consistent with the failure of readily available chemical oxidants to restore a Cu(II) species of this mutant. The structural and energetic effects of mutating the equatorial cysteine to serine, yet to be studied experimentally, are predicted to be considerable by our calculations.

Azurin↗
PubMed1985

Improved myocardial preservation by control of the oxidation-reduction potential.

Simple cold storage remains the current method of preservation of human heart allografts between removal and transplantation, but this technique is not adequate for prolonged periods of preservation. An alternative to cold storage is machine perfusion of the isolated organ. Although this technique has yielded promising results in kidney preservation, there is limited experience with the heart. One factor limiting the effectiveness of perfusion preservation is the toxicity of oxygen free radicals generated during the perfusion. The generation and reduction of these radicals results in an electron transfer, producing an electrochemical potential known as the redox potential. This study examines the role of machine perfusion in heart preservation with and without the use of a new electrochemical cell that is able to control the redox potential during the perfusion. Hearts were preserved for twenty-four hours by either simple cold storage (Group I), or by machine perfusion with redox monitoring (Group II), or redox control (Group III). Control of the oxidation-reduction potential of the perfusate during machine perfusion of isolated hearts resulted in significantly improved systolic and diastolic function after perfusion compared to hearts that underwent machine perfusion without redox control or simple cold storage.

Animals↗
PubMed2001

Structure of the M148Q mutant of rusticyanin at 1.5 A: a model for the copper site of stellacyanin.

The small blue copper protein rusticyanin from Thiobacillus ferrooxidans contains a type 1 Cu centre with a single axial ligand, Met148, which together with the His-Cys-His trigonal planar ligands produces a distorted trigonal pyramidal coordination geometry to copper. Type 1 Cu sites are found in cupredoxins and several multicopper proteins, including oxidases and nitrite reductases. The role of the axial ligand has been extensively debated in terms of its function in the fine tuning of the redox potential and spectroscopic properties of type 1 Cu sites. Numerous mutations of the Met ligand in azurins have been studied, but interpretation of the results has been complicated by the presence of the additional carbonyl oxygen ligand from Gly45, a neighbouring residue to the coordinating His46. The importance of the axial ligand has been further emphasized by the finding that the type 1 centre in Rhus vernicifera stellacyanin, with the lowest redox potential in a type 1 Cu site of 184 mV, has Gln as the axial ligand, whilst fungal laccase and ceruloplasmin, which have redox potentials of 550-800 mV, have a Leu in this position. Here, the crystal structure of the M148Q mutant of rusticyanin at 1.5 A resolution is presented. This is a significantly higher resolution than that of the structures of native rusticyanin. In addition, the M148Q structure is that of the oxidized protein while the native structures to date are of the reduced protein. The mutant protein crystallizes with two molecules per asymmetric unit, in contrast to the one present in the native crystal form. This mutant's redox potential (550 mV at pH 3.2) is lowered compared with that of the native protein ( approximately 670 mV at pH 3.2) by about 120 mV. The type 1 Cu site of M148Q closely mimics the structural characteristics of the equivalent site in non-glycosylated cucumber stellacyanin (redox potential approximately 260 mV) and, owing to the absence in rusticyanin of the fifth, carbonyl ligand present in azurin, may provide a better model for the R. vernicifera stellacyanin (redox potential approximately 184 mV) type 1 Cu site, which also lacks the fifth ligand. Furthermore, the presence of two molecules in the asymmetric unit cell indicates a potential binding region of the redox partners.

Amino Acid Substitution↗
PubMed1999

Covalent flavinylation is essential for efficient redox catalysis in vanillyl-alcohol oxidase.

By mutating the target residue of covalent flavinylation in vanillyl-alcohol oxidase, the functional role of the histidyl-FAD bond was studied. Three His(422) mutants (H422A, H422T, and H422C) were purified, which all contained tightly but noncovalently bound FAD. Steady state kinetics revealed that the mutants have retained enzyme activity, although the turnover rates have decreased by 1 order of magnitude. Stopped-flow analysis showed that the H422A mutant is still able to form a stable binary complex of reduced enzyme and a quinone methide product intermediate, a crucial step during vanillyl-alcohol oxidase-mediated catalysis. The only significant change in the catalytic cycle of the H422A mutant is a marked decrease in reduction rate. Redox potentials of both wild type and H422A vanillyl-alcohol oxidase have been determined. During reduction of H422A, a large portion of the neutral flavin semiquinone is observed. Using suitable reference dyes, the redox potentials for the two one-electron couples have been determined: -17 and -113 mV. Reduction of wild type enzyme did not result in any formation of flavin semiquinone and revealed a remarkably high redox potential of +55 mV. The marked decrease in redox potential caused by the missing covalent histidyl-FAD bond is reflected in the reduced rate of substrate-mediated flavin reduction limiting the turnover rate. Elucidation of the crystal structure of the H422A mutant established that deletion of the histidyl-FAD bond did not result in any significant structural changes. These results clearly indicate that covalent interaction of the isoalloxazine ring with the protein moiety can markedly increase the redox potential of the flavin cofactor, thereby facilitating redox catalysis. Thus, formation of a histidyl-FAD bond in specific flavoenzymes might have evolved as a way to contribute to the enhancement of their oxidative power.

Alcohol Oxidoreductases↗