Thermodynamic properties of the redox centers of Na(+)-translocating NADH:quinone oxidoreductase

Redox titration of all optically detectable prosthetic groups of Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) at pH 7.5 showed that the functionally active enzyme possesses only three titratable flavin cofactors, one noncovalently bound FAD and two covalently bound FMN residues. All three flavins undergo different redox transitions during the function of the enzyme. The noncovalently bound FAD works as a "classical" two-electron carrier with a midpoint potential (E(m)) of -200 mV. Each of the FMN residues is capable of only one-electron reduction: one from neutral flavosemiquinone to fully reduced flavin (E(m) = 20 mV) and the other from oxidized flavin to flavosemiquinone anion (E(m) = -150 mV). The lacking second half of the redox transitions for the FMNs cannot be reached under our experimental conditions and is most likely not employed in the catalytic cycle. Besides the flavins, a [2Fe-2S] cluster was shown to function in the enzyme as a one-electron carrier with an E(m) of -270 mV. The midpoint potentials of all the redox transitions determined in the enzyme were found to be independent of Na(+) concentration. Even the components that exhibit very strong retardation in the rate of their reduction by NADH at low sodium concentrations experienced no change in the E(m) values when the concentration of the coupling ion was changed 1000 times. On the basis of these data, plausible mechanisms for the translocation of transmembrane sodium ions by Na(+)-NQR are discussed.     

Adduct formation between sulfite and the flavin of phototrophic bacterial flavocytochromes c. Kinetics of sequential bleach, recolor, and rebleach of flavin as a function of pH.

The kinetics of sulfite adduct formation with the bound flavin in flavocytochromes c from the purple phototrophic bacterium Chromatium vinosum and the green phototrophic bacterium Chlorobium thiosulfatophilum have been investigated as a function of pH. Both species of flavocytochrome c rapidly react with sulfite to form a flavin sulfite adduct (k = 10(3)-10(5) M-1 s-1) which is bleached at 450-475 nm and has associated charge-transfer absorbance at 660 nm. The rate constant for adduct formation in flavocytochrome c is 2-4 orders of magnitude faster than for model flavins of comparable redox potential and is likely to be due to a basic residue near the N-1 position of the flavin, which not only raises the redox potential but also stabilizes the negatively charged adduct. There is a pK for adduct formation at 6.5, which suggests that the order of magnitude larger rate constant at pH 5 as compared to pH 10 in flavocytochrome c is due the influence of another positive charge, possibly a protonated histidine residue. The adduct is indefinitely stable at pH 5 but decomposes (the flavin recolors) in a first-order process accelerating above pH 6 (at pH 10, k = 0.1 s-1). The pK for recoloring is 8.5, which is suggestive of a cysteine sulfhydryl. On the basis of the observed pK and available chemical information, we believe that recoloring is due to a secondary effect of the reaction of sulfite with a protein cystine disulfide, which is adjacent to the flavin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of dehydrogenases/one-electron transferases by modification of flavin redox potentials. Effect of product binding on semiquinone stabilization in yeast flavocytochrome b2

Spectroscopic and potentiometric measurements have been carried out, at room temperature, during anaerobic titrations of Hansenula anomala L-lactate cytochrome c oxidoreductase (or flavocytochrome b2) both in the presence and in the absence of pyruvate (the physiological reaction product). Under the same conditions, the flavin spectral contribution was estimated and the flavosemiquinone proportion was directly determined by electron paramagnetic resonance measurements. In the present study, we show the visible light absorption and paramagnetic characteristics of the flavin radical at 18 degrees C and also the dramatic effect of pyruvate on the redox potential of each monoelectronic couple of the flavin. Thermodynamic stabilization of the semiquinone form, in the presence of pyruvate, is interpreted as a mode of regulation of flavocytochrome b2 activity. Taking into account that analogous controls have been observed with two other flavoenzymes belonging to this class of dehydrogenases/one-electron transferases, we suggest that redox potential modulation could be a type of regulation effective for the whole class of enzymes in which a semiquinone is an obligate intermediate.     

The effects of pH and semiquinone formation on the oxidation-reduction potentials of flavin mononucleotide. A reappraisal.

Calculation shows that there is poor agreement between frequently cited values for the midpoint redox potentials of the two one-electron steps in the reduction of flavin mononucleotide and equations for the lines that relate these potentials to pH and that use the published pKa values for the three redox states of the flavin [Draper, R. & Ingraham, L.L. (1969) Arch. Biochem. Biophys. 125, 802-808]. Equilibrium data for the first step in the reduction obtained by pulse radiolysis [Anderson, R.F. (1983) Biochim. Biophys. Acta 722, 158-162] show much closer agreement with theory and lead to values for the semiquinone formation constant of flavin mononucleotide that are close to those derived from measurements of the radical concentration using ESR spectroscopy. It is concluded that the data from the second method are more reliable. The redox potentials for flavin mononucleotide at pH 7.0 and 20 degrees C are calculated to be -0.207 V for the overall two-electron reduction (Em), -0.313 V for reduction of the oxidized flavin to the semiquinone (E2) and -0.101 V for the reduction of the semiquinone to the hydroquinone (E1). Information is provided to allow calculation of the three redox potentials at other pH values in the physiological range.     

One-electron oxidation-reduction properties of hepatic NADH-cytochrome b5 reductase

The one-electron oxidation-reduction properties of flavin in hepatic NADH-cytochrome b5 reductase were investigated by optical absorption spectroscopy, electron paramagnetic resonance (EPR), and potentiometric titration. An intermediate with a peak at 375 nm previously described by Iyanagi (1977) [ Iyanagi , T. (1977) Biochemistry 16, 2725-2730] was confirmed to be a red anionic semiquinone. The NAD+-bound reduced enzyme was oxidized by cytochrome b5 via the semiquinone intermediate. This indicates that electron transfer from flavin to cytochrome b5 proceeds in two successive one-electron steps. Autoxidation of the NAD+-bound reduced enzyme was slower than that of the NAD+-free reduced enzyme and was accompanied by the appearance of an EPR signal. Midpoint redox potentials of the consecutive one-electron-transfer steps in the presence of excess NAD+ were Em,1 = -88 mV and Em,2 = 147 mV at pH 7.0. This corresponds to a semiquinone formation constant of 8. The values of Em,1 and Em,2 were also studied as a function of pH. A mechanism for electron transfer from NADH to cytochrome b5 is discussed on the basis of the one-electron redox potentials of the enzyme and is compared with the electron-transfer mechanism of NADPH-cytochrome P-450 reductase.     

Structure-function relations in flavodoxins

Summary Flavodoxins are low molecular weight, FMN containing, proteins which function as electron transfer agents in a variety of microbial metabolic processes, including nitrogen fixation. Utilizing structural information obtained from x-ray crystal analysis, it has been possible to derive some new and important insights into the relationships which exist between flavin properties and protein environment by comparing the spectroscopic, thermodynamic and kinetic behavior of the flavodoxins with that of free flavin. Thus, for example, a qualitative understanding of the contribution of the protein to flavin redox potentials, semiquinone reactivity and mechanism of electron transfer is beginning to emerge. The highly negative redox potential required for the biochemical activity of the flavodoxins is accomplished by stabilizing the semiquinone via a hydrogen bond to the N-5 position of the flavin and destabilizing the fully-reduced form by constraining it to assume an unfavorable planar conformation. The reactivity of the semiquinone form is lowered by the aforementioned hydrogen bond, as well as by an interaction with a tryptophan residue in the binding site. Electron transfer is accomplished through the exposed dimethylbenzene ring of the bound coenzyme. Although it is not possible at present to determine the extent to which this understanding can be generalized to other flavoproteins, it is clear that a study of the flavodoxins will provide us with at least some of the principles which biological systems have used to modify flavin properties to fulfill a biochemical need.     

Transient kinetics of flavin-photosensitized oxidation of reduced redox proteins. Comparison of c-type cytochromes and plastocyanins.

We have used laser flash photolysis to investigate the kinetics of oxidation of reduced plastocyanins obtained from spinach and the green alga Monoraphidium braunii by the triplet states of lumiflavin, riboflavin and FMN. We have compared the results of these experiments with the kinetics of reduction of the oxidized forms of these proteins by the corresponding flavin semiquinones, as well as with the kinetics of flavin oxidation and reduction of cytochrome c552 (Class I c-type cytochrome, generic name c553) from Monoraphidium. In all cases, the rate constants for oxidation were one or two orders of magnitude larger than for reduction, consistent with the greater thermodynamic driving force for the oxidation reaction. Similar steric and electrostatic effects were observed for both reactions with all proteins, suggesting that the same (or closely adjacent) sites were being utilized for electron removal and entry. The two algal proteins were quite similar to one another in their redox properties, consistent with their physiological role of being able to substitute for one another in photosynthetic electron transport. In contrast, the algal plastocyanin was more reactive than the spinach protein in both oxidation and reduction, suggesting differences in their steric properties at the site of electron transfer.     

15N nuclear magnetic resonance of flavins.

Ninety-nine percent 15N-enriched flavins were synthesized and their proton decoupled 15N resonances were observed. The enriched compounds were [1,3-15N]riboflavin, [1,3,5-15N]riboflavin, [1,3-15N]riboflavin 5'-phosphate, [1,3,5-15N]riboflavin 5'-phosphate, and [1,3,5-15N] flavin adenine dinucleotide, [1,3,5-15N] lumiflavin, and [1,3,5-15N] lumichrome. By comparison of their spectra and from th- nuclear Overhauser effect data each 15N resonance peak could be assigned to each 15N nucleus. The order of the chemical shifts well corresponds to that of the calculated pi-electron densities. The N-3 nucleus gives the most intense inverted peak and the N-5 nucleus a small noninverted peak. By changing pH from neutral to alkaline, the chemical shift and the intensity of signal were mostly affected in the N-3 resonance of riboflavin 5'-phosphate. The N-5 signal of flavin adenine dinucleotide showed a fairly large downfield shift with the increase of temperature. These observations can be well interpreted by the chemical structure and the proposed conformation of riboflavin 5'-phosphate and flavin adenine dinucleotide.     

Flavin thermodynamics explain the oxygen insensitivity of enteric nitroreductases

Bacterial nitroreductases are NAD(P)H-dependent flavoenzymes which catalyze the oxygen-insensitive reduction of nitroaromatics, quinones, and riboflavin derivatives. Despite their broad substrate specificity, their reactivity is very specific for two-electron, not one-electron, chemistry. We now describe the thermodynamic properties of the flavin mononucleotide cofactor of Enterobacter cloacae nitroreductase (NR), determined under a variety of solution conditions. The two-electron redox midpoint potential of NR is -190 mV at pH 7.0, and both the pH dependence of the midpoint potential and the optical spectrum of the reduced enzyme indicate that the transition is from neutral oxidized flavin to anionic flavin hydroquinone. The one-electron-reduced semiquinone states of both the free enzyme and an NR-substrate analogue complex are strongly suppressed based on optical spectroscopy and electron paramagnetic resonance measurements. This can explain the oxygen insensitivity of NR and its homologues, as it makes the execution of one-electron chemistry thermodynamically unfavorable. Therefore, we have established a chemical basis for the recent finding that a nitroreductase is a member of the soxRS oxidative defense regulon in Escherichia coli [Liochev, S. I., Hausladen, A., Fridovich, I. (1999) Proc. Natl. Acad. Sci. U.S.A. 96 (7), 3537-3539]. We also report binding affinities for the FMN cofactor in all three oxidation states either determined fluorometrically or calculated using thermodynamic cycles. Thus, we provide a detailed picture of the thermodynamics underlying the unusual activity of NR.     

A comparative kinetic analysis of the reactivity of plant, horse, and human respiratory cytochrome c towards cytochrome c oxidase

Two synthetic genes coding for human and Arabidopsis cytochrome c, respectively, have been designed and constructed, and the recombinant proteins have been over-expressed in Escherichia coli cells. Thus a comparative analysis of the two heme proteins, including horse cytochrome c as a reference, has been performed. In addition to their physico-chemical properties, the redox behavior of the three proteins has been analyzed by following the kinetics of both their reduction by flavin semiquinones (lumiflavin, riboflavin, and FMN) and oxidation by cytochrome c oxidase. The resulting data indicate that the accessibility and electrostatic charge of the active site do not differ in a significant way among the three proteins, but human cytochrome c exhibits some intriguing differences when interacting with cytochrome c oxidase that could be related to the amino acid changes underwent by the latter along evolution.     

Effect of complexation of flavin radical with tryptophan on electron transfer rates: a model for flavin-protein interactions

The rates of oxidation of lumiflavin radical by ferricyanide, indole radical and oxygen are decreased by factors of four to ten as a result of complexation with tryptophan. Tyrosine, methionine and glycine were found not to measurably alter the flavin radical reactivity. Similar results were obtained using flavinyl peptides in which tryptophan or methionine were covalently linked to the flavin. These observations suggest that one of the consequences of the interaction between the flavin and a tryptophan side chain in the coenzyme binding site of the flavodoxins is to deactivate the semiquinone form of the enzyme towards oxidizing agents, thereby increasing its stability.     

Chromatium vinosum cytochrome c-552. Reduction by photoreduced flavins and intramolecular electron transfer

Cytochrome c-552 from Chromatium vinosum is an unusual heme protein in that it contains two hemes and one flavin per molecule. To investigate whether intramolecular electron transfer occurs in this protein, we have studied its reduction by external photoreduced flavin by using pulsed-laser excitation. This approach allows us to measure reduction kinetics on the mirosecond time scale. Both fully reduced lumiflavin and lumiflavin semiquinone radical reduce cytochrome c-552 with second-order rate constants of approximately 1.4 x 10(6) M-1s-1 and 1.9 x 10(8) M-1 s-1, respectively. Kinetic and spectral data and the results of similar studies with riboflavin indicate that both the flavin and heme moieties of cytochrome c-552 are reduced simultaneously on a millisecond time scale, with the transient formation of a protein-bound flavin anion radical. This is suggested to be due to rapid intramolecular electron transfer. Further, steric restrictions play an important role in the reduction reaction. Studies were conducted on the redox processes following photolysis of CO-ferrocytochrome c-552 in which the flavin was partly oxidized to resolve the kinetics of electron transfer between the heme and flavin of cytochrome c-552. Based on these results, we conclude that intramolecular electron transfer from ferrous heme to oxidized flavin occurs with a first-order rate constant of greater than 1.4 x 10(6) s-1.     

Modification of redox equilibria between heme and flavin within yeast flavocytochrome b2 (l-lactate cytochrome c reductase) upon binding of pyruvate,the reaction product

Direct determinations of the concentration of semiquinone spin in redox equilibrium with the cytochrome b₂ moiety were carried out at room temperature in the presence of added pyruvate or in its absence. Results show that redox potentials of the one-electron couples of the prosthetic flavin are markedly affected by binding of pyruvate the reaction product in the oxidation of l-lactate. The proportion of flavin semiquinone nearly reaches then 100 per cent.     

Reactivity of hydrogen sulfide with peroxynitrite and other oxidants of biological interest

Hydrogen sulfide (H(2)S) is an endogenously generated gas that can also be administered exogenously. It modulates physiological functions and has reported cytoprotective effects. To evaluate a possible antioxidant role, we investigated the reactivity of hydrogen sulfide with several one- and two-electron oxidants. The rate constant of the direct reaction with peroxynitrite was (4.8±1.4)×10(3)M(-1) s(-1) (pH 7.4, 37°C). At low hydrogen sulfide concentrations, oxidation by peroxynitrite led to oxygen consumption, consistent with a one-electron oxidation that initiated a radical chain reaction. Accordingly, pulse radiolysis studies indicated that hydrogen sulfide reacted with nitrogen dioxide at (3.0±0.3)×10(6)M(-1) s(-1) at pH 6 and (1.2±0.1)×10(7)M(-1) s(-1) at pH 7.5 (25°C). The reactions of hydrogen sulfide with hydrogen peroxide, hypochlorite, and taurine chloramine had rate constants of 0.73±0.03, (8±3)×10(7), and 303±27M(-1) s(-1), respectively (pH 7.4, 37°C). The reactivity of hydrogen sulfide was compared to that of low-molecular-weight thiols such as cysteine and glutathione. Considering the low tissue concentrations of endogenous hydrogen sulfide, direct reactions with oxidants probably cannot completely account for its protective effects.     

Redox activation of galactose oxidase: thin-layer electrochemical study

The redox activation of galactose oxidase by various oxidants is characterized by using a unique thin-layer electrochemical cell. Activation is shown to be strictly a redox process and can be rapidly accomplished by using ferricyanide, cobalt terpyridine or tetracyanomonophenanthroline ferrate, and a control electrode to control solution potential. This oxidation is a one-electron process and does not result in modified galactose oxidase which exhibits enhanced activity. On the contrary, this oxidation is required for activity. The solution potential dependence of activity is independent of which of these mediator-titrants is used, the concentration used, and which of various substrates is used in the determination. The substrates used were acetol, dihydroxyacetone, glycerin, 2-propyn-1-ol, allyl alcohol, 2-butyne-1,4-diol, furfuryl alcohol, benzyl alcohol, 4-pyridylcarbinol, galactose, and stachyose. The E1/2 and n values obtained are 0.40 +/- 0.005 V vs. SHE and 0.9 +/- 0.1 electron at pH 7.3. E1/2 is defined as the potential at which half the maximal enzymatic activity is observed and probably reflects the E0' of the enzymic group involved in activation. A model is proposed in which activation occurs during turnover due to the redox buffering (by oxidants) of an enzymic Cu(II)/Cu(I) state which has a higher E0' than in resting galactose oxidase. The pH dependence of E1/2 is 60 mV/pH unit in the pH range 6.0-8.0. The data suggest that the deprotonation of an amino acid residue facilitates the one-electron oxidation (activation) of galactose oxidase.     

Kinetics of oxidation of serotonin by myeloperoxidase compounds I and II.

The oxidation of serotonin (5-hydroxytryptamine) by the myeloperoxidase intermediates compounds I and II was investigated by using transient-state spectral and kinetic measurements at 25.0 +/- 0.1 degrees C. Rapid scan spectra demonstrated that both compound I and compound II oxidize serotonin via one-electron processes. Rate constants for these reactions were determined using both sequential-mixing and single-mixing stopped-flow techniques. The second order rate constant obtained for the one-electron reduction of compound I to compound II by serotonin is (1.7 +/- 0.1) x 10(7) M(-1) x s(-1), and that for compound II reduction to native enzyme is (1.4 +/- 0.1) x 10(6) M(-1) x s(-1) at pH 7.0. The maximum pH of the compound I reaction with serotonin occurs in the pH range 7.0-7.5. At neutral pH, the rate constant for myeloperoxidase compound I reacting with serotonin is an order of magnitude larger than for its reaction with chloride, (2.2 +/- 0.2) x 10(6) M(-1) x s(-1). A direct competition of serotonin with chloride for myeloperoxidase compound I oxidation was observed. Our results suggest that serotonin may have a role to protect lipoproteins from oxidation and to prevent enzymes from inactivation caused by the potent oxidants HOCl and active oxygen species.     

Laser flash photolysis as a probe of redox protein-membrane interactions: effect of binding of spinach plastocyanin and horse cytochrome c to lipid bilayer vesicles on the kinetics of reduction by flavin semiquinone

Spinach plastocyanin binds to both electrically neutral and positively charged lipid bilayer vesicles, whereas cytochrome c only binds electrostatically to negatively charged vesicles. Laser flash photolysis using lumiflavin semiquinone as a reductant demonstrates that the reactivity of plastocyanin is increased as much as 6-fold when it is membrane bound whereas the rate constant for cytochrome c reduction is decreased by approximately a factor of 3. Membrane-bound plastocyanin reduction occurs via a two-step mechanism, probably involving prior association of lumiflavin semiquinone with the bilayer. In contrast, cytochrome c reduction in the membrane-bound state follows simple second-order kinetics, implying that the redox site in the bound state is still accessible to lumiflavin semiquinone in solution, although the rate constant is decreased by approximately 3-fold. These results are interpreted as indicating that the bilayer-protein interaction with plastocyanin leads to a steric blockage of the electron-transfer site from the aqueous phase. Little or no hindrance of the redox site occurs with cytochrome c, suggesting a high degree of mobility of this protein on the bilayer surface. Although the increase in plastocyanin reactivity upon binding to the bilayer is quite interesting, its cause remains unclear and requires further study. The results illustrate the utility of laser flash photolysis as a probe of membrane-protein interactions.     

Why do proteins use selenocysteine instead of cysteine?

Selenocysteine is present in a variety of proteins and catalyzes the oxidation of thiols to disulfides and the reduction of disulfides to thiols. Here, we compare the kinetic and thermodynamic properties of cysteine with its selenium-containing analogon, selenocysteine. Reactions of simple selenols at pH 7 are up to four orders of magnitude faster than their sulfur analogs, depending on reaction type. In redox-related proteins, the use of selenium instead of sulfur can be used to tune electrode, or redox, potentials. Selenocysteine could also have a protective effect in proteins because its one-electron oxidized product, the selanyl radical, is not oxidizing enough to modify or destroy proteins, whereas the cysteine-thiyl radical can do this very rapidly.     

Effect of pH on oxidation-reduction potentials of 8 alpha-N-imidazole-substituted flavins

The pKa values for the various ionic forms of 8 alpha-N-imidazolylriboflavin were determined in its oxidized and hydroquinone forms and estimated for its semiquinone form. The pH dependence of the absorption and fluorescence spectral properties and potentiometric titration data show the pKa values for the oxidized form to be 6.02 +/- 0.03 for the 8 alpha-imidazole nitrogen and 9.67 +/- 0.05 for the N(3) position of the flavin ring. The pH dependence of the oxidation-reduction potential was determined by spectrocoulometric titrations, and the data points were compared with computer-simulated plots. Two pKa values for the hydroquinone form of the flavin were determined and assigned. The pKa for the imidazole ring is found to be 6.9 +/- 0.1 and for the N(1) position of the flavin hydroquinone is found to be 5.5 +/- 0.1. Analysis of the pH dependence of the one-electron couples E2 (flavoquinone/flavin semiquinone, Flox/Fl.) and E1 (flavin semiquinone/flavin hydroquinone, Fl./Flred) resulted in an estimated pKa of 6.5 for the 8 alpha-imidazole ring in the flavin semiquinone form. These data show the possible involvement of the ionization of the 8 alpha-imidazole substituent in the redox chemistry of flavoenzymes containing either an 8 alpha-N1- or an 8 alpha-N3-histidyl-linked covalent flavin coenzyme. Future work on oxidation-reduction potentials of this class of enzymes must take into consideration the influence of the 8 alpha-histidyl substituent.     

On the environment and the rotational motion of amphiphilic flavins in artificial membrane vesicles as studied by electron paramagnetic resonance spectroscopy

This paper continues the studies of vesicle-bound flavins (‘anisotropic flavin chemistry’). It is possible to anchor the flavin nucleus in various modes within the lipid/water interface by means of long aliphatic chains and using different saturated lipids, thereby mimicking the specific binding of the coenzyme to the apoprotein in flavoproteins. Based on absorption spectroscopy and EPR spectroscopy studies we explored the rotational mobility and the microenvironment of membrane-bound amphiflavin radicals. N(5)-unsubstiluted amphiflavin radicals exhibit a similarly high disproportionation constant as known from isotropic flavin chemistry. However, reasonable stabilization of the radical was achieved by introduction of an alkyl group in position 5 in the reduced state prior to the one-electron oxidation. Adopting the fine structure of the corresponding EPR spectra as assay for the mobility of the semiquinone, we determined rotational relaxation times ranging from 60 ns in the crystalline state down to 10 or 15 ns in the liquid-crystalline state of the membrane. The solvatochromic effect shown by absorption spectra of the membrane-bound flavin radicals reflects a dielectric constant of the microenvironment of ? = 30–40, corresponding to the lipid/water interface region. The results obtained in this study are consistent with those obtained previously, from fluorescence analyses, supporting our former conclusions.