Relevance of the flavin binding to the stability and folding of engineered cholesterol oxidase containing noncovalently bound FAD

Abstract

The flavoprotein cholesterol oxidase (CO) from Brevibacterium sterolicum is a monomeric flavoenzyme containing one molecule of FAD cofactor covalently linked to His69. The elimination of the covalent link following the His69Ala substitution was demonstrated to result in a significant decrease in activity, in the midpoint redox potential of the flavin, and in stability with respect to the wild-type enzyme, but does not modify the overall structure of the enzyme. We used CO as a model system to dissect the changes due to the elimination of the covalent link between the flavin and the protein (by comparing the wild-type and H69A CO holoproteins) with those due to the elimination of the cofactor (by comparing the holo- and apoprotein forms of H69A CO). The apoprotein of H69A CO lacks the characteristic tertiary structure of the holoprotein and displays larger hydrophobic surfaces; its urea-induced unfolding does not occur by a simple two-state mechanism and is largely nonreversible. Minor alterations in the flavin binding region are evident between the native and the refolded proteins, and are likely responsible for the low refolding yield observed. A model for the equilibrium unfolding of H69A CO that also takes into consideration the effects of cofactor binding and dissociation, and thus may be of general significance in terms of the relationships between cofactor uptake and folding in flavoproteins, is presented.

Figure 1.

Comparison of spectral properties of holoenzyme (solid line) and apoprotein (dashed line) of H69A CO. (A) Protein fluorescence (excitation at 280 nm, 0.1 mg protein/mL); (B) Near-UV CD spectrum (0.5 mg protein/mL). (Inset) Far-UV CD spectrum (0.2 mg protein/mL); (C) ANS fluorescence (excitation at 370 nm; 1.6 μM protein [0.1 mg/mL] added of 560 μM ANS). Proteins were in 50 mM potassium phosphate, pH 7.5; measurements were performed at 15°C.

Figure 2.

Equilibrium denaturation curves of holo- (circles, solid lines) and apoprotein (squares, dashed lines) of H69A CO detected by using different techniques. (A,B) Tryptophan fluorescence (excitation at 280 nm, 0.1 mg protein/mL): The fraction of unfolded CO was determined from the fluorescence intensity at ∼340 nm using the value for the untreated and 8-M urea-treated proteins as reference (A); position of the emission peak maximum (B). (C) Near-UV CD measurements at 291 nm (proteins were 0.5 mg/mL). Lines represent the best fit obtained using a two-state denaturation model (Tanford 1968; Pace 1990; Caldinelli et al. 2005). For all experiments, CO samples were equilibrated at 15°C in 50 mM potassium phosphate, pH 7.5, containing the given urea concentration. The reported values have been corrected for readings prior to protein addition.

Figure 3.

Results of titration of holo- (A) and apoprotein (B) of H69A CO with ANS, as a function of urea concentration. ΔF (filled symbols): maximal change in fluorescence intensity at 500 nm at saturating ANS concentration; K_d (open symbols) apparent dissociation constant for ANS binding. (C) Ratio of the ΔF and K_d values determined at different urea concentrations for the holo- (●) and apoprotein (■) of H69A, and for the wild-type CO (▲). Protein samples (0.1 mg/mL) were equilibrated for 60 min at 15°C in 50 mM potassium phosphate, pH 7.5, at the given urea concentration. Following each addition of ANS to the protein–urea mixtures, fluorescence spectra were recorded from 450 to 600 nm with excitation at 370 nm.

Figure 4.

Effect of urea on the ANS fluorescence in the presence of holo- (A, circles) and apoprotein (B, squares) of H69A CO. Fluorescence intensity at 500 nm (filled symbols) and emission maximum (open symbols) were determined on CO samples (0.1 mg protein/mL) equilibrated for 60 min at 15°C in the presence of increasing urea concentrations in 50 mM potassium phosphate, pH 7.5. Fluorescence spectra were recorded with excitation at 370 nm after the addition of 0.1 mM ANS. The reported values have been corrected for the emission of the solution prior to ANS addition.

Figure 5.

Temperature dependence of spectroscopic signals for the holo- (●) and apoprotein (■) of H69A CO: (A) tryptophan fluorescence; (B) ANS fluorescence; (C) far-UV CD. All experiments were performed in 50 mM potassium phosphate, pH 7.5. Protein concentration was 0.1 mg/mL in all fluorescence measurements, but was higher for far-UV CD (0.25 mg/mL). When required, ANS was added at 0.5 mM final concentration. Spectral signals were monitored continuously during progressive heating from 20°C to 80°C at a heating rate of 0.5°C/min, and are given as percent of the total observed change regardless of the direction of the change. Inset of B: DSC trace for apo-H69A (dashed line), heating rate 0.5°C/min (a similar result was obtained at heating rate 0.1°C/min, data not shown). Protein was 1.5 mg/mL in 100 mM Tris-HCl, pH 8.5. The continuous solid line is a fit performed according to single step equilibrium transition model (ΔH = 270 kJ mol⁻¹, T_d = 35.5°C used as fitting parameters).

Figure 6.

Effect of 2 M urea on release (A,B) and binding (C) of flavin cofactor to H69A CO. (A) Time course of protein (filled symbols) and flavin (open symbols) fluorescence released by H69A CO (0.2 mg protein/mL) in the absence (circle) and in the presence (square) of 2 M urea. (B) The samples obtained as reported in (A) were incubated in a buffer containing 2.5 M KBr and the same concentration of urea as above. (C) Time course of protein fluorescence intensity at 340 nm during the FAD binding (1.3 μM) to apo-H69A (0.12 μM, 0.0075 mg/mL) in the absence (circle) and in the presence (square) of 2 M urea. All the experiments were performed at 15°C in 50 mM potassium phosphate, pH 7.5.

Figure 7.

Comparison of the protein fluorescence spectrum of holo- (1) and apoprotein (2) of H69A CO with that obtained following the reconstitution of apoprotein with FAD (3,4). Reconstituted H69A CO holoenzyme obtained starting from 6 M urea-treated apoprotein and addition of 10-molar excess of FAD simultaneously to 10-fold dilution of denaturant (3) or after the 10-fold dilution of denaturant for 1 h at 15°C (4).

Scheme 1.

A schematic representation of the structural changes of holoenzyme and apoprotein of H69A COs ensuing from increasing concentrations of urea. The arrows indicate the sites of trypsin cleavage.