Y

-nh h3c

Fig. 1. The structures of biologically relevant flavin species in three different redox states.

resistance against proteolysis. Moreover, we have demonstrated that the covalent bond can raise the oxidative power of the flavin.11

Flavins are capable of performing both one- and two-electron transfer processes. The redox potentials for the two-electron reductions of FAD and FMN free in solution at pH 7.0 are —219 mV12 and —205 mV,13 respectively. Because a small percentage of the flavin semiquinone (one-electron reduced) is formed in thermodynamic equilibrium with the quinone (oxidized) and hydroquinone (two-electron reduced) (Fig. 1), the overall two-electron reduction can be analyzed for the two single-electron transfer steps, and a redox potential for both one-electron reductions can be determined. The semiquinone species can exist in either the blue neutral form (absorbance maximum in the 500- to 600-nm region) or the red anionic form (absorbance maximum in the 370- to 400-nm region), depending on the pH (pK = 8.3) (Fig. 1). When bound to apoprotein the flavin redox characteristics can change dramatically. Some flavoenzymes show essentially no stabilization of semiquinone on reduction, whereas others give nearly 100% stabilization. In the latter case, the protein may stabilize the neutral semiquinone over a wide pH range,

11 M. W. Fraaije, R. H. H. van den Heuvel, W. J. H. van Berkel, and A. Mattevi, J. Biol. Chem. 274, 35514 (1999).

12 H. J. Lowe and W. M. Clarke, J. Biol. Chem. 221, 983 (1956).

13 R. D. Draper and L. L. Ingraham, Arch. Biochem. Biophys. 125, 802 (1968).

indicative of a high pK value, whereas others stabilize only the anionic form, indicative of a low pK. When the semiquinone is stabilized the redox potentials of the two steps in reduction are more separated.14

At present, the redox potentials of about 40 flavoenzymes are known, varying from —367 mV for nitroalkane oxidase15 to +55 mV for vanillyl-alcohol oxidase (VAO).11 For a selected number of flavoproteins the effects of substrate binding and pH have been studied16 to obtain a better understanding of the catalytic mechanism. Especially when a crystal structure and active site mutant enzymes are available, such studies can reveal important information about the regulation of the flavin redox potential. As an example, we describe the redox properties of VAO, a covalent flavoprotein with an exceptionally high redox potential. However, we first briefly review the most common methods of flavin redox potential determination.

Determination of Flavin Redox Potentials

Electrochemical Methods

Electrochemical methods have proved to be effective approaches for studying the redox properties of flavoenzymes.17'18 In these methods the oxidation state of the enzyme is changed electrochemically, using an electrode, while measuring the change in some property of the enzyme-bound flavin responsive to its redox state. The redox state of the flavin can be monitored, for example, by absorption spectroscopy, cyclic voltammetry, circular dichroism, or electron spin resonance. However, as the three different redox states of flavoenzymes have distinct spectral properties, absorption spectroscopy is most often used.

The electrochemical technique also allows the determination of the total number of electrons transferred from the electrode to the flavin. The power of this method lies in the fact that it gives quantitative information, which is especially useful when analyzing multi-redox-center enzymes.

Spectrophotometric Method

In the spectrophotometric method the equilibrium of partially reduced enzyme-bound flavin and a redox reference dye is analyzed optically. This is perhaps the simplest method for determining the redox potential of a flavoprotein because

14 W. M. Clark, in "Oxidation-Reduction Potentials of Organic Systems." Williams & Wilkins, Baltimore, MD, 1960.

15 G. Gadda and P. F. Fitzpatrick, Biochemistry 37,6154 (1998).

16 M. T. Stankovich, in "Chemistry and Biochemistry of Flavoenzymes" (F. Mueller, ed.), Vol. I, p. 401. CRC Press, Boca Raton, FL, 1991.

18 M. T. Stankovich, Anal. Biochem. 109, 295 (1980).

there is no need for any specialized equipment. This optical method only requires a procedure to introduce reducing equivalents into the solution, a system to make a cuvette anaerobic, and a thermostatted scanning spectrophotometer.

In addition to the discussed electrochemical technique, several other procedures are available for introducing reducing equivalents into the reaction system. Chemical reduction by sodium dithionite is widely used for this purpose, as dithion-ite is a strong reducing agent.19 Major disadvantages of dithionite, however, are its high reactivity with molecular oxygen, its changes in redox potential with pH, and its instability at low pH values (pH < 6.5). Moreover, the oxidized form of dithionite can form an adduct with several flavoproteins.20 Photoreduction with 5-deazaflavin is another procedure for introducing reducing equivalents in a system. Catalysis of 5-deazaflavin in photoreduction is due to the formation of the strongly reducing 5-deazaflavosemiquinone species, which is generated by photolysis of the preformed 5-deazaflavin dimer.21 Photoreduction is technically less complex to use than chemical reduction, because after the initial addition of 5-deazaflavin, reducing equivalents will be introduced continuously after exposing the system to light. A major drawback is that 5-deazaflavin is not commercially available and other photocatalysts do not always reduce the enzyme-bound flavins completely. A third commonly used method for introducing reducing equivalents into a system is the xanthine-xanthine oxidase method.22 This method is extremely convenient for the determination of redox potentials of many flavoproteins. It uses xanthine oxidase, a low-potential reference dye, and xanthine to introduce reducing equivalents. We discuss this procedure in more detail, as we used it to determine the redox properties of VAO.

Xanthine-Xanthine Oxidase Method

Xanthine oxidase is a flavin-containing enzyme, which oxidizes xanthine to urate with the subsequent reduction of molecular oxygen to hydrogen peroxide. Xanthine oxidase can use, besides molecular oxygen, a number of low-potential dyes as electron acceptors, such as benzyl viologen (Em = —359 mV at pH 7.0) and methyl viologen (Em = —449 mV at pH 7.0). These dyes can react with other electron acceptors and also with most enzyme-bound flavins.

The assay described here is based on the method described by Massey.22 Equal concentrations of the flavoprotein and the reference dye, typically 8-10 (J.M, 400 (jlM xanthine, and 2.5 fiM methyl viologen or benzyl viologen, are placed

20 V. Massey, F. Muller, R. Feldberg, M. Schuman, P. A. Sullivan, L. G. Howell, S. G. Mayhew, R. G. Matthews, and G. P. Foust, J. Biol. Chem. 244, 3999 (1969).

21 V. Massey, M. Stankovich, and P. Hemmerich, Biochemistry 17, 1 (1978).

22 V. Massey, in "Flavins, and Flavoproteins 1990" (B. Curti, S. Ronchi, and G. Zanetti, eds.), p. 59. Walter de Gruyter, Berlin, 1991.

in an anaerobic cuvette (total volume, 800 /xl). The use of a Hellma (Plainview, NY) QS-117-104 cuvette sealed with a Subaseal 13 septum gives excellent results. A 10 mM xanthine stock solution is prepared by mixing xanthine with water and adjusting the pH to pH 11 with potassium hydroxide. The mixture of flavoprotein, reference dye, and xanthine is made anaerobic by either continuous flushing with oxygen-free argon gas or repeated cycles of evacuation and flushing with oxygen-free argon. The reduction of the flavoprotein and the reference dye is initiated by the anaerobic addition of xanthine oxidase, using a Hamilton syringe. Anaerobic conditions during the reductive process are maintained by continuously flushing the headspace of the cuvette with oxygen-free argon. To ensure equilibration between the oxidized and reduced species of enzyme and reference dye, the reduction must be sufficiently slow. The rate of reduction can be regulated by the concentration of xanthine oxidase. The final concentration needed depends on the redox potentials of the enzyme-bound flavin and dye, but is generally within 0.5-2.0 /ig/ml. During the xanthine oxidase-mediated reduction, typically lasting 1-2 hr for full reduction of both enzyme and dye, spectra are recorded every 30 sec with a scanning diode-array spectrophotometer at 25°. Ideally, the concentrations of the oxidized and reduced forms of the flavoprotein and the reference dye are determined at a wavelength at which the other redox partner has an isosbestic point between the oxidized and reduced forms or has no absorbance at all. It is important to note that the redox potential of the reference dye should be within 30 mV of the flavoprotein to obtain reliable data. A number of useful redox reference dyes are given in Durst et alP and Mayhew.24 By using this method we could estimate the redox potentials of VAO variants, usually within 5 mV.

The potentials at 50% reduction of the flavoenzyme can be calculated by the Nernst equation14:

£h(dye) = £m(dye) + 2303(RT/ndyeF)log(dyeox/dyered)

£h(E) = EJE) + 2303(RT/nEF)\og(Em/Ered) Z?h(dye) = £h(E) (at equilibrium)

where £h is the observed potential, Em is the potential when the concentrations of oxidized and reduced forms are equal, R is the gas constant (8.31 J K_1 moP1), T is the temperature in degrees Kelvin, n is the number of electrons needed to convert the oxidized form to the reduced form, and F is the Faraday constant (96,496 J V"1 mol"1). Thus, at 25°, 2303(RT/nF) is equal to 0.059/n V.

23 R. A. Durst, E. A. Blubaugh, K. A. Bunding, M. L. Fultz, W. A. MacCrehan, and W. T. Yap, Clin.

24 S. G. Mayhew, in "Flavoprotein Protocols" (S. K. Chapman and G. A. Reid, eds.), Vol. 131, p. 49.

Humana Press, Totowa, NJ, 1999.

Redox Properties of Vanillyl-Alcohol Oxidase

Vanillyl-Alcohol Oxidase

Vanillyl-alcohol oxidase (VAO; EC 1.1.3.38) is a homooctameric flavo-enzyme25 that catalyzes the oxidative demethylation of methyl ethers of p-cresol, providing the ascomycete Penicillium simplicissimum with a tool for metabolizing these lignin-derived aromatic compounds.26 Each VAO subunit contains an 8a-/V3-histidyl-FAD as covalently bound prosthetic group.25 The crystal structure of VAO has revealed that each VAO monomer consists of two domains: the larger domain comprises residues 1-270 and 500-560 and forms the FAD-binding domain, whereas residues 271-499 form the smaller cap domain.27 The catalytic center of VAO is located at the interface of the two domains in the core of the protein. From sequence alignments it was recognized that VAO is a representative of a widespread family of structurally related oxidoreductases sharing a conserved flavin-binding domain.28 A similarly folded domain is, for example, also present in the peripheral membrane respiratory flavoenzyme D-lactate dehydrogenase29 and in UDP-A^acetylenolpyruvylglucosamine reductase (MurB), a flavoenzyme involved in peptidoglycan biosynthesis.30

Redox catalysis of VAO involves two half-reactions, in which the flavin is reduced by the substrate (reduction) and subsequently the reduced flavin is re-oxidized by molecular oxygen (oxidation). The reductive half-reaction involves the initial transfer of a hydride from the substrate to the flavin, resulting in a binary complex between the two-electron reduced enzyme and the /^-quinone methide of the substrate. In the oxidative part of the reaction, the reduced flavin is reoxidized by molecular oxygen with the concomitant hydration of the quinone methide intermediate (Fig. 2). For the reaction with the physiological substrate 4-(methoxymethyl)phenol, the reductive half-reaction is rate limiting in overall catalysis.31

Redox Properties

The midpoint redox potential of ligand-free VAO was determined by the xanthine-xanthine oxidase method in the presence of the redox reference dye

25 E. de Jong, W. J. H. van Berkel, R. P. van der Zwan, and J. A. M. de Bont, Eur. J. Biochem. 208,651 (1992).

26 M. W. Fraaije, M. Pikkemaat, and W. J. H. van Berkel, Appl. Environ. Microbiol. 63,435 (1997).

27 A. Mattevi, M. W. Fraaije, A. Mozzarelli, L. Olivi, A. Coda, and W. J. H. van Berkel, Struct. Fold. Des. 5,907(1997).

28 M. W. Fraaije, W. J. H. van Berkel, J. A. E. Benen, J. Visser, and A. Mattevi, Trends Biochem. Sci. 23, 206 (1998).

29 O. Dym, E. A. Pratt, C. Ho, and D. Eisenberg, Proc. Natl. Acad. Sci. U.S.A. 97, 9413 (2000).

30 T. E. Benson, D. J. Filman, C. T. Walsh, and J. M. Hogle, Nat. Struct. Biol. 2, 644 (1995).

31 M. W. Fraaije and W. J. H. van Berkel, J. Biol. Chem. 272,18111 (1997).

o2 H2O2

Fig. 2. Oxidative demethylation of 4-(methoxymethyl)phenol by VAO.

o2 H2O2

Fig. 2. Oxidative demethylation of 4-(methoxymethyl)phenol by VAO.

thionin (Em — +60 mV) at pH 7.5. Figure 3 clearly shows that the enzyme-bound flavin and the dye reduce simultaneously via a single two-electron process, indicating that the dye and the flavin have a similar redox potential. The observed two-electron process fits well with the catalytic function of VAO, which involves the initial hydride transfer from the substrate to the flavin. When log(VA0ox/VAOred) versus log(dyeox/dyered) was plotted according to Minnaert32 a midpoint redox potential of +55 mV was estimated (see inset, Fig. 3).33 This redox potential is exceptionally high compared with other flavin-dependent enzymes. Only thiamine oxidase, also containing an 8a-iV3-histidyl-FAD, has an equally high redox potential (+55 mV).34 The redox potential of VAO complexed with the substrate analog isoeugenol was determined in the presence of methylene blue (£m =+11 mV). The enzyme reduced in a single two-electron reduction process and the redox potential was estimated to be +15 mV. Thus, when the active site cavity is saturated with the phenolic ligand isoeugenol the midpoint redox potential decreases by 40 mV.

In our studies we mainly focused on the rationale for the exceptionally high redox potential of VAO. A recurrent feature on analyzing flavin-containing oxi-doreductases is the presence of a hydrogen bond donor near the N5 atom of the flavin prosthetic group. In most oxidoreductases this hydrogen bond donor is a backbone or a side-chain nitrogen atom.35 However, in VAO such a hydrogen bond donor is absent. Instead, a negatively charged residue, Asp-170, is located near the flavin N5 atom (distance, 3.5 A) (Fig. 4).27 The side chain of Asp-170 is positioned in such a way that during catalysis it might interact with the reduced FAD cofactor, thereby stabilizing the reduced form. Intriguingly, glycolate oxidase also lacks a hydrogen bond donor to flavin N5 and, like VAO, this enzyme has a relatively high redox potential (Em = —21 mV).36

32 K. Minnaert, Biochim. Biophys. Acta 110,42 (1965).

33 R. H. H. van den Heuvel, M. W. Fraaije, A. Mattevi, and W. J. H. van Berkel, J. Biol. Chem. 275, 14799 (2000).

34 C. Gomez-Moreno, M. Choy, and D. E. Edmondson, J. Biol. Chem. 254, 7630 (1979).

35 M. W. Fraaije and A. Mattevi, Trends Biochem. Sci. 25, 126 (2000).

36 C. Pace and M. Stankovich, Biochemistry 25, 2516 (1986).

FIG. 3. Determination of the redox potential of wild-type VAO. VAO, 9 /iM in potassium phosphate buffer, pH 7.5 at 25°, was reduced in the presence of 9 fiM thionin by the xanthine-xanthine oxidase method. The reduction was finished after 100 min. Inset: log(VA0ox/VAOre<i) (measured at 439 nm after correction for thionin) versus log(dyeox/dyered) (measured at 600 nm). [Data from R. H. H. van den Heuvel, M. W. Fraaije, A. Mattevi, and W. J. H. van Berkel, J. Biol. Chem. 275,14799 (2000).]

Wavelength (nm)

FIG. 3. Determination of the redox potential of wild-type VAO. VAO, 9 /iM in potassium phosphate buffer, pH 7.5 at 25°, was reduced in the presence of 9 fiM thionin by the xanthine-xanthine oxidase method. The reduction was finished after 100 min. Inset: log(VA0ox/VAOre<i) (measured at 439 nm after correction for thionin) versus log(dyeox/dyered) (measured at 600 nm). [Data from R. H. H. van den Heuvel, M. W. Fraaije, A. Mattevi, and W. J. H. van Berkel, J. Biol. Chem. 275,14799 (2000).]

The role of Asp-170 in VAO has been studied in detail by site-directed mutagenesis. When Asp-170 is replaced by glutamate or serine the flavin redox potential decreases 50 and 160 mV, respectively.33 Kinetic characterization of the mutants revealed that Asp-170 is required for rapid substrate-mediated flavin reduction. D170E and D170S are 50-fold and 1000-fold less active, respectively.33 Moreover, structural analysis of D170S suggested that the mutation does not induce any significant structural perturbations. These results indicate that both the presence

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