Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase

Abstract

The FAD-dependent choline oxidase has a flavin cofactor covalently attached to the protein via histidine 99 through an 8α-N(3)-histidyl linkage. The enzyme catalyzes the four-electron oxidation of choline to glycine betaine, forming betaine aldehyde as an enzyme-bound intermediate. The variant form of choline oxidase in which the histidine residue has been replaced with asparagine was used to investigate the contribution of the 8α-N(3)-histidyl linkage of FAD to the protein toward the reaction catalyzed by the enzyme. Decreases of 10-fold and 30-fold in the k_cat/K_m and k_cat values were observed as compared with wild-type choline oxidase at pH 10 and 25 °C, with no significant effect on k_cat/K_O using choline as substrate. Both the k_cat/K_m and k_cat values increased with increasing pH to limiting values at high pH consistent with the participation of an unprotonated group in the reductive half-reaction and the overall turnover of the enzyme. The pH independence of both ^D(k_cat/K_m) and ^Dk_cat, with average values of 9.2 ± 3.3 and 7.4 ± 0.5, respectively, is consistent with absence of external forward and reverse commitments to catalysis, and the chemical step of CH bond cleavage being rate-limiting for both the reductive half-reaction and the overall enzyme turnover. The temperature dependence of the ^Dk_red values suggests disruption of the preorganization in the asparagine variant enzyme. Altogether, the data presented in this study are consistent with the FAD-histidyl covalent linkage being important for the optimal positioning of the hydride ion donor and acceptor in the tunneling reaction catalyzed by choline oxidase.

A number of enzymes, including dehydrogenases (1–3), monooxygenases (4–7), halogenases (8–11), and oxidases (7, 12, 13), employ flavin cofactors (FAD or FMN) for their catalytic processes. About a tenth of all flavoproteins have been shown to contain a covalently attached cofactor, which may be linked at the C8M position via histidyl, tyrosyl, or cysteinyl side chains or at the C6M position via a cysteinyl side chain (14). Glucooligosaccharide oxidase (15, 16), hexose oxidase (17), and berberine bridge enzyme (18, 19) are examples of flavoproteins (FAD as cofactor) with both linkages present in one flavin molecule. The covalent linkages in flavin-dependent enzymes have been shown to stabilize protein structure (20–22), prevent loss of loosely bound flavin cofactors (23), modulate the redox potential of the flavin microenvironment (20, 23–27), facilitate electron transfer reactions (28), and contribute to substrate binding as in the case of the cysteinyl linkage (20). However, no study has implicated a mechanistic role of the flavin covalent linkages in enzymatic reactions in which a hydride ion is transferred by quantum mechanical tunneling.

The discovery of quantum mechanical tunneling in enzymatic reactions, in which hydrogen atoms, protons, and hydride ions are transferred, has attracted considerable interest in enzyme studies geared toward understanding the mechanisms underlying the several orders of magnitudes in the rate enhancements of protein-catalyzed reactions compared with non-enzymatic ones. Tunneling mechanisms have been shown in a wide array of cofactor-dependent enzymes, including flavoenzymes. Examples of flavoenzymes in which the tunneling mechanisms have been demonstrated include morphinone reductase (29, 30), pentaerythritol tetranitrate reductase (29), glucose oxidase (31–33), and choline oxidase (34). Mechanistic data on Class 2 dihydroorotate dehydrogenases, also with a flavin cofactor (FMN) covalently linked to the protein moiety (35, 36), could only propose a mechanism that is either stepwise or concerted with significant quantum mechanical tunneling for the hydride transfer from C6 and the deprotonation at C5 in the oxidation of dihydroorotate to orotate (37). This leaves choline oxidase as the only characterized enzyme with a covalently attached flavin cofactor (12, 38), where the oxidation of its substrate occurs unequivocally by quantum mechanical tunneling.

Choline oxidase from Arthrobacter globiformis catalyzes the two-step FAD-dependent oxidation of the primary alcohol substrate choline to glycine betaine with betaine aldehyde, which is predominantly bound to the enzyme and forms a gem-diol species, as intermediate (Scheme 1). Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism against stress conditions, thus making genetic engineering of relevant plants of economic interest (39–45), and the biosynthetic pathway for the osmolyte is a potential drug target in human microbial infections of clinical interest (46–48). The first oxidation step catalyzed by choline oxidase involves the transfer of a hydride ion from a deprotonated choline to the protein-bound flavin followed by reaction of the anionic flavin hydroquinone with molecular oxygen to regenerate the oxidized FAD (for a recent review see Ref. 50). The gem-diol choline, i.e. hydrated betaine aldehyde, is the substrate for the second oxidation step (49), suggesting that the reaction may follow a similar mechanism. The isoalloxazine ring of the flavin cofactor, which is buried within the protein, is physically constrained through a covalent linkage via the C(8) methyl of the flavin and the N(3) atom of the histidine side chain at position 99 (Fig. 1) (12). Also contributing to the physical constrain are the proximity of Ile-103 to the pyrimidine ring and the interactions of the backbone atoms of residues His-99 through Ile-103 with the isoalloxazine ring. The rigid positioning of the isoalloxazine ring could only permit a solvent-excluded cavity of ∼125 ų adjacent to the re face of the FAD to accommodate a 93-ų choline molecule in the substrate binding domain (12). Mechanistic data thus far obtained on choline oxidase, coupled with the crystal structure of the wild-type enzyme resolved to 1.86 Å, are consistent with a quantum tunneling mechanism for the hydride ion transfer occurring within a highly preorganized enzyme-substrate complex (Scheme 2) (12, 34, 50). Exploitation of the tunneling mechanism requires minimal independent movement of the hydride ion donor and acceptor, with the only dynamic motions permitted being the ones that promote the hydride transfer reaction.

SCHEME 1.

Two-step, four-electron oxidation of choline catalyzed by choline oxidase.

FIGURE 1.

x-ray crystal structure of the active site of wild-type choline oxidase resolved to 1.86 Å (PDB 2jbv). Note the significant distortion of the flavin ring at the C(4a) atom, which is due to the presence of a C(4a) adduct (69).

SCHEME 2.

The hydride ion transfer reaction from the α-carbon of the activated choline alkoxide species to the N(5) atom of the isoalloxazine ring of the enzyme-bound flavin in choline oxidase.

In the present study, the contribution of the physically constrained flavin isoalloxazine ring to the reaction catalyzed by choline oxidase has been investigated in a variant enzyme in which the histidine residue at position 99 was replaced with an asparagine. The results suggest that, although not being required per se, the covalent linkage in choline oxidase contributes to the hydride tunneling reaction by either preventing independent movement or contributing to the optimal positioning of the flavin acting as hydride ion acceptor with respect to the alkoxide species acting as a donor. However, the covalent linkage is not required for the reaction.

EXPERIMENTAL PROCEDURES

Materials

Recombinant wild-type choline oxidase gene (pET/codAmg) in a permanent stock Escherichia coli strain XLI Blue was used as template for site-directed mutagenesis. E. coli strain Rosetta (DE3)pLysS was from Novagen (Madison, WI). QIA prep Spin Miniprep kit was from Qiagen (Valencia, CA). A QuikChange site-directed mutagenesis kit was from Stratagene. Oligonucleotides for site-directed mutagenesis and sequencing of the mutant gene were from Sigma Genosys (The Woodlands, TX). Choline chloride was from ICN (Aurora, OH). 1,2-[²H₄]Choline bromide was from Isotech Inc. (Miamisburg, OH). Glucose and glucose oxidase were from Sigma-Aldrich. All other reagents used were of the highest purity commercially available.

Site-directed Mutagenesis, Expression, and Purification of CHO-H99N

The mutant gene for the choline oxidase variant containing asparagine at position 99, CHO-H99N,² was prepared using the pET/codAmg gene for the wild-type enzyme as template (38) with forward and reverse oligonucleotides as primers in site-directed mutagenesis. The site-directed mutagenesis was performed using the QuikChange kit, following the manufacturer's manual. DMSO (2% final) was added to enhance separation of the template DNA upon denaturation. The mutation was confirmed by sequencing the resultant gene at the DNA Core Facility at Georgia State University using an Applied Biosystems Big Dye Kit on an Applied Biosystems model ABI377 DNA sequencer. The plasmid with the mutant gene (pET/codAmg H99N) was used to transform E. coli strain Rosetta(DE3)pLysS by electroporation and permanent stocks of the transformed cells prepared and stored at −80 °C. The H99N variant of choline oxidase was expressed and purified to homogeneity with the incorporation of 10% glycerol throughout the purification procedures following the previously described purification protocol (12, 38, 51).

Biochemical Characterization of CHO-H99N

To determine whether the flavin cofactor in CHO-H99N was covalently attached to the protein, the variant enzyme was treated with 10% trichloroacetic acid as previously described for CHO-E312D (12) with modifications. The extinction coefficient of the bound flavin in CHO-H99N was determined by concurrently treating free FAD with 10% trichloroacetic acid and subjecting the acidified free FAD to the same experimental conditions as the mutant enzyme. The extinction coefficient of the acidified free FAD was used as reference to determine the extinction coefficient of the CHO-H99N-bound FAD.

Enzyme Assays

Enzyme activities of CHO-H99N were measured by the method of initial rates in 50 mm sodium phosphate or 50 mm sodium pyrophosphate as described previously for the wild-type enzyme (52) by monitoring the rate of oxygen consumption with a computer-interfaced Oxy-32 oxygen monitoring system (Hansatech Instrument Inc.) at 25 °C with choline or 1,2-[²H₄]choline as substrate.

Steady-state kinetic parameters for CHO-H99N were determined at varying concentrations of choline (0.01–20 mm) and oxygen (0.2–1 mm) at pH 10. The desired oxygen concentration for each assay was obtained by bubbling the appropriate O₂/N₂ gas mixture for a minimum of 10 min to equilibrate the reaction mixture. pH profiles of the steady-state kinetic parameters were obtained in the pH range from 6 to 11. All the enzyme assays for the pH profiles were conducted in 50 mm sodium pyrophosphate. Substrate kinetic isotope effect on the steady-state kinetic parameters were determined by alternating varying concentrations of choline and 1,2-[²H₄]choline at atmospheric concentration of oxygen in the pH range of 6.5 and 10.5 at 0.5-pH unit intervals.

Rapid kinetics at varying concentrations of choline or 1,2-[²H₄]choline was carried out with a Hi-Tech SF-61 stopped-flow spectrophotometer thermostatted from 10 to 28 °C at ∼2 °C intervals. The rates of flavin reduction were monitored by measuring the decrease in absorbance at 450 nm upon mixing the oxidized enzyme with the organic substrate, both prepared in 50 mm sodium pyrophosphate, pH 9. The stopped-flow apparatus was previously made anaerobic by treating with a mixture of glucose (5 mm) and glucose oxidase (∼30 units/ml) in 100 mm sodium pyrophosphate, pH 6, overnight to scrub oxygen and rinsed with oxygen-free 50 mm sodium pyrophosphate, pH 9. The enzyme and substrate were made anaerobic as previously described (53). Equal volumes of CHO-H99N and choline (or 1,2-[²H₄]choline) were mixed anaerobically in the stopped-flow spectrophotometer resulting in a reaction mixture with a final enzyme concentration of ∼10 μm and substrate concentrations of 0.05 to 10 mm, with each substrate concentration assayed in triplicate (differences in each set of three observed rates were <5%). The substrate isotopomers were alternated to determine the isotopic effect on the rate constant for flavin reduction at different temperatures.

Data Analysis

Data were fit with KaleidaGraph (Synergy Software, Reading, PA) and EnzFitter softwares (Biosoft, Cambridge, UK). Steady-state kinetic parameters were determined by fitting the initial rates data to Equation 1 which describes a sequential steady-state kinetic mechanism where e represents the concentration of enzyme, k_cat is the turnover number of the enzyme at saturating substrates concentrations, and K_a and K_b represent the Michaelis constants for the organic substrate (A) and oxygen (B), respectively. The pH dependences of the steady-state kinetic parameters were determined by fitting the initial rates data to Equation 2, which describes a curve with a slope of +1 and a plateau region at high pH, where C is the pH-independent value of the kinetic parameter. Stopped-flow traces were fit to Equation 3, which describes a single exponential process, where k_obs represents the observed rate of flavin reduction, t is time, A_t is the value of absorbance at 450 nm at any given time, A is the amplitude for the total change in absorbance, and A_∞ is the absorbance at infinite time. Pre-steady-state kinetic parameters were determined by fitting the observed rates data to Equation 4, where k_obs is the observed rate for the reduction of the enzyme bound flavin, k_red is the limiting rate of flavin reduction at saturating substrate concentrations and K_d is the dissociation constant for binding of the substrate to the enzyme. The temperature dependence of the rate of flavin reduction was determined by fitting the stopped-flow data with Eyring's equation (Equation 5), where k_B is Boltzmann constant, h is Plank's constant, k_red is the limiting rate of flavin reduction at saturating substrate concentrations, R is gas constant, T is temperature, and ΔS^‡ and ΔH^‡ are the entropy and enthalpy of activations, respectively. The entropy of activation is calculated from the y-intercept, and the enthalpy of activation is calculated from the slope of the Eyring plot. The temperature dependence of the kinetic isotope effect on the rate of flavin reduction was determined by fitting the data with Arrhenius equation (Equation 6), where KIE is the deuterium kinetic isotope effect on the rate of flavin reduction, A_H/A_D is the isotope effect on the pre-exponential factors, and [E_a(D) − E_a(H)] is the isotope effect on the energy of activation.

RESULTS

Purification and Biochemical Characterization of CHO-H99N

The choline oxidase variant in which histidine 99, which participates in a covalent linkage with the C8M atom of FAD, was replaced with asparagine was expressed and purified following the same protocol previously described for wild-type choline oxidase (38) with the addition of 10% glycerol to all the purification solutions. Unlike the wild-type (38, 51, 54) and other choline oxidase variant enzymes (12, 55, 56), which contain a mixture of oxidized flavin and air-stable, anionic flavosemiquinone upon purification, the H99N mutant enzyme was purified with the bound flavin cofactor in the fully oxidized state, as indicated by the UV-visible absorbance spectrum showing absorbance maxima at 390 and 450 nm (supplemental Fig. S1). The UV-visible absorbance spectrum of the supernatant after treatment of the variant enzyme with 10% trichloroacetic acid and centrifugation to remove partially denatured protein showed the presence of FAD, consistent with the flavin cofactor not being covalently bound to the protein upon substituting histidine 99 with asparagine. An extinction coefficient of 13.9 mm⁻¹ cm⁻¹ was determined at 450 nm for CHO-H99N, as compared with 11.4 mm⁻¹ cm⁻¹ for the wild-type choline oxidase (54). The steady-state kinetic parameters with choline as substrate for CHO-H99N under atmospheric oxygen conditions are shown in Table 1, along with the parameters previously determined for the wild-type enzyme. Both the ^appk_cat and ^app(k_cat/K_m) values, as well as the specific activity of the mutant enzyme, were significantly lower than in the wild-type enzyme, suggesting that protein flavinylation is important for the alcohol oxidation reaction catalyzed by choline oxidase.

TABLE 1.

Comparison of specific activities and apparent steady-state kinetic parameters of CHO-H99N with wild-type choline oxidase

Total protein amounts were determined by the Bradford method (70). Specific activity was measured in atmospheric oxygen with 10 mm choline as substrate in 50 mm potassium phosphate, pH 7, and 25 °C. For the kinetic parameters, the enzymatic activities were measured at varying concentrations of choline in the range from 0.005 to 10 mm, in 50 mm potassium phosphate, pH 7, and 25 °C, using fully oxidized enzymes.

Enzyme	Specific activity	app(kcat/Km)	appkcat	appKm
	units/mg	m−1s−1	s−1	mm
CHO-H99N	0.28	790	1.03	1.30
CHO-WTa	8.0	25,000	15.0	0.60

^a Data are from Ref. (12).

Steady-state Kinetic Mechanism

The steady-state kinetic parameters for the asparagine 99 mutant enzyme were determined at pH 10 and 25 °C by measuring the initial rate of oxygen consumption at varying concentrations of choline and oxygen. The data were best fit with the sequential steady-state kinetic equation (Equation 1), suggesting that replacement of histidine 99 with asparagine does not alter the order of substrate binding and product release with respect to the wild-type enzyme. CHO-H99N catalyzes the oxidation of choline to glycine betaine with a 10-fold decrease in the k_cat/K_m value, a 30-fold decrease in the k_cat value, but with no significant difference in the k_cat/K_O value (Table 2). These kinetic data suggest that the covalent linkage between His-99 N(3) and the FAD C8M atom is important for the reductive half-reaction and the overall turnover of the enzyme, but it is not involved in the reaction of the reduced flavin with oxygen.

TABLE 2.

Comparison of steady-state kinetic parameters for CHO-H99N and wild-type choline oxidase with choline as substrate

Steady-state kinetic parameters were determined at varying concentrations of choline and oxygen in 50 mm sodium pyrophosphate, pH 10, and 25 °C.

Kinetic parameters	CHO-H99N	CHO-WTa
kcat, s−1	1.86 ± 0.01	60 ± 1
kcat/Km, m−1s−1	22,360 ± 780	237,000 ± 9000
Km, mm	0.08 ± 0.01	0.25 ± 0.01
KO2, mm	0.015 ± 0.001	0.69 ± 0.03
kcat/KO2, m−1s−1	123,730 ± 4,400	86,400 ± 3,600
D(kcat/Km)b	9.2 ± 3.3	10.7 ± 2.6
Dkcatb	7.4 ± 0.5	7.3 ± 1.0

^a Data are from Ref. (52).

^b The pH-independent values are reported here.

Effect of pH on the Reductive and Oxidative Half-reactions

The pH profiles of the steady-state kinetic parameters for CHO-H99N were carried out in the pH range from 6 to 11 at 25 °C at varying concentrations of both choline and oxygen. The k_cat/K_m and k_cat values increased with increasing pH to limiting values at high pH (Fig. 2), consistent with the requirement of an unprotonated group for catalysis in both the reductive half-reaction and the overall turnover of the variant enzyme. Apparent pK_a values of 8.2 ± 0.1 and 7.1 ± 0.1 were determined from the k_cat/K_m and k_cat pH profiles, respectively (Fig. 2 and supplemental Table S1). There was no pH effect on the k_cat/K_O values with an average pH-independent limiting value of 82,500 ± 32,000 m⁻¹ s⁻¹ in the pH range from 7 to 11 (Fig. 2 and supplemental Table S1). This value is not significantly different from the pH-independent value of 86,400 m⁻¹ s⁻¹ that was previously reported for the wild-type enzyme (34), consistent with the oxidative half-reaction not being affected in the CHO-H99N variant enzyme. An average K_O value of 24 μm was estimated in the pH range experimented (supplemental Table S1), indicating that the H99N enzyme is at least 90% saturated with oxygen when the initial rates of reaction are measured at atmospheric oxygen concentrations, i.e. 250 μm.

FIGURE 2.

pH dependence of the k_cat/K_m (●), k_cat/K_oxygen (□), and k_cat (○) values for CHO-H99N with choline as substrates at 25 °C. Enzymaticd activities were measured at varying concentrations of choline (0.01–20 mm) and oxygen (0.2–1 mm). Data for k_cat/K_m and k_cat were fit to Equation 2, and data for k_cat/K_oxygen were fit to y = 4.9.

Substrate Kinetic Isotope Effects

The substrate kinetic isotope effects on the k_cat/K_m and k_cat values were determined with 1,2-[²H₄]choline at 25 °C under air-saturated buffer conditions, ensuring at least 90% saturation of the enzyme with oxygen due to the low K_O values (see above). The k_cat/K_m values for both choline and 1,2-[²H₄]choline yielded similar pK_a values of 8.4 ± 0.1 (supplemental Fig. S2) and resulted in a pH-independent ^D(k_cat/K_m) value of 9.2 ± 3.3 (Fig. 3) in the pH range of 6.5 and 10.5. These results are consistent with the pK_a value of ∼8.4 being the thermodynamic pK_a for the unprotonated group that participates in the reductive half-reaction catalyzed by the enzyme, and with lack of external forward and reverse commitments to catalysis (57–59). The pH dependence of the k_cat values for both choline and 1,2-[²H₄]choline also yielded similar pK_a values of ≤7.0 (supplemental Fig. S2), resulting in a pH-independent ^Dk_cat value of 7.4 ± 0.5 (Fig. 3). The kinetic data for the pH dependence of the kinetic isotope effects of CHO-H99N are summarized in supplemental Table S2.

FIGURE 3.

pH dependence of the ^D(k_cat/K_m) and ^Dk_cat values for CHO-H99N. Data were fit to y = 0.94 in A and to y = 0.87 in B. Enzymatic activities were measured at varying concentrations of choline and 1,2-[²H₄]choline at saturating oxygen concentration under atmospheric conditions.

Effect of Temperature on the Rate Constant for Flavin Reduction

The effect of temperature on the rate constant for flavin reduction (k_red) and the associate kinetic isotope effects (^Dk_red) were investigated to probe the hydride transfer reaction catalyzed by CHO-H99N. The rates of flavin reduction were determined under anaerobic conditions at varying concentrations of choline or 1,2-[²H₄]choline from 10 to 28 °C in 50 mm sodium pyrophosphate, pH 9, using a stopped-flow spectrophotometer. The limited range of temperatures stems from the instability of the CHO-H99N enzyme at temperatures ≥30 °C over the prolonged times required for data acquisition. All the stopped-flow traces showed single exponential processes of flavin reduction with both choline and 1,2-[²H₄]choline, as illustrated in Fig. 4 for the traces at 16 °C with 10 mm choline (a) and 1,2-[²H₄]choline (b). In all cases, the UV-visible absorbance spectrum of the reduced flavin showed a well defined maximum at 365 nm, consistent with the presence of the anionic hydroquinone species at the end of the reduction reaction (Fig. 4 illustrates the data at 16 °C). The observed rates of flavin reduction were fit to Equation 4 to obtain the k_red values for choline and 1,2-[²H₄]choline, and the associated kinetic isotope effects (see Fig. 4 for the data at 16 °C and supplemental Fig. S3 for all other temperatures). The k_red and ^Dk_red values obtained in the temperature range from 10 to 28 °C are summarized in supplemental Table S3.

FIGURE 4.

Anaerobic reductions of CHO-H99N with choline and 1,2-[²H₄]choline at 16. 1 °C and pH 9.0. A, stopped-flow traces for choline (a) and 1,2-[²H₄]choline (b) at 10 mm substrate concentration. Data were fit to Equation 5. Time indicated is after the end of flow, i.e. 2.2 ms. B, rates of flavin reduction as a function of substrate concentration with choline (●) and 1,2-[²H₄]choline (○). Data were fit to Equation 6. C, UV-visible absorbance spectrum of reduced flavin bound to CHO-H99N after mixing with 10 mm choline.

The k_red values for choline and 1,2-[²H₄]choline increased monotonically with increasing temperature yielding different slopes (Fig. 5), as analyzed according to Eyring's theory. This resulted in a temperature dependence of the ^Dk_red values (Fig. 5), with values ranging from 8.05 ± 0.13 at 28 °C to 9.17 ± 0.12 at 10 °C. Different enthalpies of activation (ΔH^‡) were determined for the cleavages of the CH and CD bonds (Table 3). A large isotope effect with a finite value was determined for the energy of activation for the reaction (ΔE_a = (E_a)_H − (E_a)_D) (Table 3). Finally, a value that was close to unity was estimated for the isotope effect on the pre-exponential factors (A_H′/A_D′) calculated from the ratio of the y-intercepts of the temperature-dependent Eyring plots for choline and 1,2-[²H₄]choline (Table 3). All taken together, these thermodynamic data indicate that the hydride transfer reaction in the H99N enzyme is significantly different from that of the wild-type enzyme for which previous results showed a large and temperature-independent kinetic isotope effect on the reductive half-reaction, similar ΔH^‡ values for the cleavage of the CH and CD bonds of choline, a negligible ΔE_a value, and a large A_H′/A_D′ ratio (Table 3).

FIGURE 5.

Temperature dependence of the k_red and ^D(k_red) values for CHO-H99N with choline or 1,2-[²H₄]choline from 10 to 28 °C. A, Eyring plot of the k_red values versus temperature with choline (●) and 1,2-[²H₄]choline (○) as substrate. Data were fit to Equation 5. Standard deviations associated with the measurements are ≤5% of the measured value. B, Arrhenius plot of the ^D(k_red) values versus temperature. Data were fit to Equation 6.

TABLE 3.

Comparison of the thermodynamic parameters for the reductive half-reactions catalyzed by the H99N and wild-type choline oxidase

Parameter	CHO-H99Na	CHO-WTb
	kred	kcat/Km
ΔH‡H,c kJ mol−1	23 ± 1	18 ± 2
ΔH‡D,c kJ mol−1	29 ± 1	18 ± 5
−TΔS‡H, kJ mol−1	44 ± 4	24 ± 2
−TΔS‡D, kJ mol−1	44 ± 3	30 ± 5
ΔS‡H,d kJ K−1 mol−1	0.15 ± 0.01	0.08 ± 0.01
ΔS‡D,d kJ K−1 mol−1	0.15 ± 0.01	0.10 ± 0.02
ΔG‡H,d kJ mol−1	67 ± 4	42 ± 3
ΔG‡D,d kJ mol−1	73 ± 3	48 ± 7
ΔEa, kJ mol−1	6 ± 1	0.4 ± 4.2
AH′/AD′e	0.9 ± 0.1	14 ± 3
KIEf	T-dependent	T-independent

^a Conditions: rates of flavin reduction were measured in 50 mm sodium pyrophosphate, pH 9.0, at different temperatures under anaerobic conditions.

^b Data are from Ref. (34).

^c Data were calculated by using the Eyring equation (Equation 5).

^d Data for 25 °C.

^e A_H′/A_D′ is the ratio of the y-intercepts obtained by fitting the kinetic data with choline and 1,2-[²H₄]choline to the Eyring equation (Equation 5).

^f KIE is the substrate kinetic isotope effect associated with the cleavage of the CH and CD bonds of choline in the reaction-catalyzed y choline oxidase.

DISCUSSION

Previous studies established that the reaction of alcohol oxidation in choline oxidase occurs through the transfer of a hydride ion from the α-carbon of choline alkoxide to the N(5) atom of the FAD cofactor within a highly preorganized enzyme-substrate complex (for a recent review see Ref. 50). In such a preorganized complex, the activated alcohol substrate, and the isoalloxazine of the flavin are allowed minimal independent movement, because the former establishes electrostatic interactions at its opposite ends with the charged residues Glu-312 and His-466 and the latter is rigidly positioned by way of a covalent linkage (Fig. 1) to the protein moiety and several interactions with backbone atoms of amino acid residues at positions 100–103. In this study, the contribution of the covalent linkage between the C8M of the flavin and the N(3) atom of the histidine side chain at position 99 to the alcohol oxidation reaction in choline oxidase have been investigated using a variant form of the enzyme in which the histidine residue was replaced with an asparagine. In common with the wild-type enzyme, the histidine to asparagine variant form of choline oxidase displayed a steady-state kinetic mechanism with oxygen reacting with the reduced flavin before release of the organic product of the reaction, the requirement of an unprotonated group acting as a base in the reductive half-reaction of choline oxidation, the chemical step of CH bond cleavage being rate-limiting in both the reductive half-reaction and the overall enzyme turnover, and the lack of observable ionizable groups that participate in the oxidative half-reaction in which the reduced flavin reacts with oxygen. These data collectively suggest that the enzyme containing asparagine at position 99 maintains an overall integrity that is similar to that of the wild-type enzyme, and thereby mechanistic differences between the two enzymes can be attributed to the presence or absence of the covalent flavin attachment to the protein. This conclusion is strongly reinforced by the observation that the second-order rate constant for reaction of the reduced flavin with oxygen (k_cat/K_oxygen) is essentially the same with values of ∼85,000 m⁻¹ s⁻¹ in the mutant and wild-type enzymes, because one would expect such a critical reaction of flavin oxidation to occur at significantly different rates had the protein integrity of the mutant enzyme been different from that of the wild-type enzyme.

The FAD C8M covalent linkage with histidine 99 in choline oxidase is important for the reductive half-reaction in which choline is oxidized to betaine aldehyde with concomitant hydride ion transfer to the flavin but is not involved in the oxidative half-reaction in which the flavin is oxidized with concomitant formation of hydrogen peroxide. Evidence supporting the importance of the covalent linkage to the hydride transfer reaction is the ∼45-fold decrease in the rate constant for anaerobic flavin reduction (k_red) with choline as substrate at 25 °C with respect to the wild-type enzyme, along with the 10-fold and 30-fold decreases in the k_cat/K_m and k_cat values with choline as substrate, and the lack of significant changes in the k_cat/K_oxygen value for the H99N enzyme as compared with the wild-type form of choline oxidase at pH 10 and 25 °C. Similar results were observed upon replacing either one of the other two histidine residues that are located in the active site of choline oxidase, namely His-351 (56) and His-466 (55). Thus, while His-99 (see below), His-351, and His-466 participate with different roles in the oxidation of choline to yield betaine aldehyde, none of them provide the electrostatic stabilization that has been shown in glucose oxidase (60, 61) to be required for the reaction of flavin oxidation. In this regard, previous results with a substrate analog devoid of positive charge demonstrated that it is the positive charge harbored on the trimethylamine group of the enzyme-bound betaine aldehyde that plays an important role for oxygen reactivity in choline oxidase (49, 51). In agreement with the lack of involvement of ionizable groups in the oxidative half-reaction is the observation that no pK_a values in the range between 7.0 and 11.0 were detected in the pH profile of the k_cat/K_oxygen value with the H99N enzyme, as also was previously reported for the H351A and H466A forms of choline oxidase (55, 56).

The FAD-histidyl covalent linkage is important for the optimal positioning of the flavin in the enzyme-alkoxide complex that is required for the environmentally assisted tunneling of the hydride ion in the reaction catalyzed by choline oxidase. Evidence for this conclusion comes from the comparison of the effects of temperature on the rates of anaerobic flavin reduction (k_red) and the related kinetic isotope effects (^Dk_red) determined with a stopped-flow spectrophotometer for the H99N enzyme (this study) and the wild-type form of choline oxidase (34). Indeed, the reaction of hydride ion transfer in the wild-type enzyme with the flavin covalently attached to the protein has been previously shown to occur via quantum mechanical tunneling within a highly preorganized active site environment (34, 62). Such a mechanism of hydride ion transfer is manifested mechanistically in the large kinetic isotope effects on the Eyring pre-exponential factors (A_H′/A_D′), temperature-independent substrate kinetic isotope effects, negligible isotope effects on the energies of activation (ΔE_a = (E_a)_H − (E_a)_D), and finite and indistinguishable enthalpies of activation (ΔH^‡) for protium and deuterium transfers (34, 62). In contrast, the reaction of hydride ion transfer in the H99N enzyme with the flavin non-covalently attached to the protein requires significant sampling of the reactive configuration that is conducive to the tunneling reaction, as shown by the A_H′/A_D′ ratio not being significantly different from unity, the large value for the ΔE_a with protium and deuterium, the effect of temperature on the ^Dk_red values with 1,2-[²H₄]choline, and the different ΔH^‡ values for the cleavage of the CH and CD bonds of the substrate (63) (Table 3). Such a drastic change in the mechanism of hydride transfer most likely arises from either an increased conformational freedom or a rearrangement of the position of the flavin isoalloxazine ring acting as the hydride ion acceptor with respect to the choline alkoxide acting as the hydride donor, which necessarily results from the lack of the flavin covalent attachment to the protein moiety.³ This, in turn, establishes that the covalent linkage in flavin-dependent enzymes, besides stabilizing protein structure (20–22), preventing the loss of loosely bound flavin cofactors (23), modulating the redox potential of the flavin (20, 23–27), and facilitating electron transfer reactions (28) as amply demonstrated from studies of enzymes other than choline oxidase, may also be important for the optimal positioning of the flavin in those enzymes where hydride ions are transferred through tunneling mechanisms.

The chemical step of hydride ion transfer is rate limiting in the reaction of alcohol oxidation catalyzed by the choline oxidase form with the non-covalently attached flavin. This conclusion stems from the large and pH-independent substrate kinetic isotope effects on the reductive half-reaction (k_cat/K_m) and the overall turnover (k_cat) of the H99N variant enzyme with 1,2-[²H₄]choline as substrate under steady-state conditions. The enzyme with the non-covalent flavin is similar to the wild-type enzyme containing covalent flavin, in that the wild-type enzyme was also previously shown to lack external forward and reverse commitments to catalysis (52, 64). Independent evidence supporting the lack of commitments to catalysis in the Asn-99 variant enzyme is the lack of perturbation in the kinetic pK_a value of 8.4 ± 0.1 determined in the pH profile for the k_cat/K_m value when choline is substituted with 1,2-[²H₄]choline as substrate. Indeed, for a substrate such as 1,2-[²H₄]choline that is about 9-fold slower than choline, a ΔpK_a of up to one would be expected if the CH bond cleavage were not fully rate limiting in the reductive half-reaction where choline is oxidized to betaine aldehyde (65).

Although His-99 is not the active site base that participates in catalysis by abstracting the hydroxyl proton of choline to activate the substrate for the hydride transfer to the flavin, it is important for the overall polarity of the active site. Evidence for these conclusions comes from the pH profiles of the steady-state kinetic parameters k_cat/K_m and k_cat showing that an unprotonated group with thermodynamic pK_a value of 8.2 to 8.4 is required for catalysis in the H99N enzyme, as was previously established for the wild-type enzyme where the thermodynamic pK_a value was ∼7.5 (52, 64). In this respect, the H99N enzyme behaves in a fashion similar to that displayed in choline oxidase variants where the other two active site histidine residues were each replaced with alanine. In those cases, a catalytic base was shown to be required for the reductive half-reaction with thermodynamic pK_a values of ∼8.0 for the H351A enzyme (56) and 9.0 for the H466A enzyme (55). Consequently, it appears that all of the histidine residues located in the active site of choline oxidase modulate to different extents the polarity of the active site, which is essential for the efficient transfer of the hydroxyl proton of the alcohol substrate to an as yet unidentified base in the active site of the enzyme that triggers the subsequent hydride transfer reaction in which choline is oxidized.

The histidyl covalent attachment of the flavin to the protein moiety is required for the unusual stabilization of the anionic flavosemiquinone of choline oxidase toward molecular oxygen. Indeed, the wild-type form of choline oxidase is purified with the enzyme-bound flavin as a mixture of oxidized and anionic flavosemiquinone (38, 51, 54), and extensive incubation at pH 6.0 is necessary to fully oxidize the flavin (54). In contrast, throughout the purification process of the H99N enzyme the enzyme-bound flavin is fully oxidized. Thus far this is the first instance among several enzymatically active variants of choline oxidase where active site residues were selectively replaced with other amino acids, such as E312A, E312D, E312Q (12), H351A (56), V464T, V464A (53), and H466A⁴ (55), in which lack of air stabilization of the flavosemiquinone has been observed. Although these results suggest an important role for protein flavinylation in the lack of reactivity of the semiquinone with oxygen, more studies will have to be carried out to elucidate the mechanism at a molecular level.

In conclusion, the results of the mechanistic study on the variant form of choline oxidase, in which the covalent linkage between the isoalloxazine ring of the FAD and the histidine residue at position 99 is removed, show the importance of the flavin C8M to N(3) histidyl linkage for the optimal positioning of the flavin in the reaction of alcohol oxidation catalyzed by the enzyme. Although the enzyme with non-covalent flavin maintains mechanistic properties that are similar to those of the wild-type enzyme with covalently attached flavin, the highly preorganized active site configuration that is required for the tunneling of the hydride ion is disrupted through the lack of a covalent linkage of the flavin to the protein. This study thereby shows that protein flavinylation, besides stabilizing protein structure, preventing the loss of loosely bound flavin cofactors, and modulating the redox properties of the flavin as demonstrated in other studies, may also be important for the optimal positioning of the flavin in those enzymes where hydride transfer reactions utilize tunneling.