Highly UV-Absorbing Complex in Selenomethionine-Substituted Alcohol Dehydrogenase from Sulfolobus solfataricus Antonietta Giordano,† Consiglia Russo,† Carlo A. Raia,†,* Irina M. Kuznetsova,‡ Olga V. Stepanenko,‡ and Kostantin K. Turoverov*,‡ Institute of Protein Biochemistry, C. N. R., Via G. Marconi 10, I-80125 Naples, Italy and Institute of Cytology of the Russian Academy of Science, Tikhoretsky av. 4, St. Petersburg 194064, Russia Received December 28, 2003
The aim of this work was to explain the previously discovered effect of significant decrease in intrinsic fluorescence intensity of SsADH caused by replacement of S atoms of methionine residues to Se (Giordano, A.; Raia, C. A. J. Fluorescence 2003, 13, 17-24) on the basis of the analysis of its 3D structure. It was found that all selenium atoms are located far from both Trp95 and Trp117 and could not cause their fluorescence quenching. At the same time, it was determined that substitution of S by Se causes enhanced protein absorption in the UV-region. This effect was explained by the formation of Se complex with some groups of protein. It was revealed that this complex does not participate in fluorescence and does not transfer excitation energy to tryptophan or tyrosine residues. Keywords: alcohol dehydrogenase • sulfur/selenium fluorescence quenching • nonradiative energy transfer • tryptophan fluorescence
3D structure. The role of tyrosine residues in the formation of SsADH fluorescence properties is especially emphasized.
Introduction Alcohol dehydrogenase from the thermoacidophilic sulfurdependent crenarchaeon Sulfolobus solfataricus (SsADH) is a thermophilic NAD+-dependent homotetrameric zinc enzyme whose crystal structure has recently been determined at 1.85 Å resolution by using a selenomethionine-substituted enzyme (file 1JVB.ent1,2). Cloning and overexpression in E. coli of the SsADH gene, as well as the kinetic and thermostability properties of the enzyme have been reviewed.3 SsADH is a dimer of dimers; each monomer is formed by a polypeptide chain of 347 amino acids (37 588 Da) comprising two domains separated by a cleft and containing two zinc atoms with a catalytic and a structural role, respectively. The subunit contains two tryptophan residues (Trp95 and Trp117), both located in the catalytic domain, and 13 tyrosine residues, 8 of which are also located in the catalytic domain, and the other 5 are located in coenzyme-binding domain. The main features of SsADH fluorescence have been considered by Giordano and Raia.4 The present work is the development of the previous studies with the aim to explain the blue fluorescence spectrum of this enzyme, to evaluate the contribution of individual tryptophan and tyrosine residues in the bulk fluorescence of protein, and finally, to explain a significant quenching of SsADH fluorescence evoked by the change of sulfur atom of methionine residues to selenium atoms on the basis of the analysis of its * To whom correspondence should be addressed. E-mail: kkt@ mail.cytspb.rssi.ru. Phone: +7-812-247-1957. Fax: +7-812-247-0341. Carlo A. Raia, IBP, CNR, Via G. Marconi 10, 80125 Naples, Italy. Fax: +39-0817257240. E-mail:
[email protected]. † Institute of Protein Biochemistry. ‡ Institute of Cytology of the Russian Academy of Science. 10.1021/pr034132d CCC: $27.50
2004 American Chemical Society
Materials and Methods Chemicals. L-tryptophan and L-tyrosine were from Aldrich (San Diego, CA). Pyrazole, L-methionine, and Se-L-methionine were from Sigma (St. Louis, MO). NAD+ was purchased from Applichem (Darmstadt, FRG). Solutions of NAD+ were prepared in water and adjusted to approximately pH 6.5 with diluted NaOH. Other chemicals were A grade substances from Sigma or Applichem. All solutions were made up with MilliQ water. Protein Purification. The native and selenomethionine recombinant wild-type SsADH were prepared according to the method described in refs 1 and 3, respectively. The mass spectra of the selenomethionyl protein confirmed 96% substitution Met f Se-Met.1 Both homogeneous natural and Seprotein were exhaustively dialyzed against 20 mM Tris-HCl, pH 8.5 in the absence and the presence of 10 mM 2-mercaptoethanol, respectively, for removal of endogenous coenzyme as described in ref 3, and then were stored at -20 °C in the respective buffers. Protein concentration was routinely determined with a Bio-Rad protein assay kit, using bovine serum albumin as standard. As judged by quantitative amino acid analysis,5 the concentration values obtained from the colorimetric measurement are overestimated by 30%, so that they have to be normalized to effective values. The correction factor used is in excellent agreement with that calculated by spectrophotometric3 or spectrofluorimetric titration of the activesite concentration of SsADH with NAD+ in the presence of excess pyrazole. For the present study, titration of coenzymefree SsADH and Se-SsADH (typically 0.1-0.3 µM tetramer, in the presence of 50 mM glycine/NaOH, pH 9.8, 5 mM pyrazole) Journal of Proteome Research 2004, 3, 613-620
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was performed monitoring the fluorescence quenching at λmax of 320 nm (λex ) 280 nm) occurring on NAD+ addition. The active-site concentration determined by the intersection of the titration plot resulted nearly identical to that deduced from the colorimetric method. Fluorescence Studies. Steady-state emission and excitation spectra were recorded with a JASCO FP-777 spectrofluorometer, using a quartz cuvette containing 500 µL of solution and thermostated with a temperature regulated cell holder. The buffer used was 50 mM glycine-NaOH, pH 9.8. For the emission spectra (λex ) 280, 295 nm) the protein concentration was 0.028 mg/mL (0.19 µM tetramer) and the excitation and emission slit bandwidths were 10 and 5 nm, respectively. For the excitation spectra (λem ) 320, 365 nm) the protein concentration was 0.025 mg/mL (0.16 µM tetramer) and the excitation and emission slit bandwidths were 3 and 5 nm, respectively. UV spectra were recorded with a Cary 1E spectrophotometer equipped with a computer-controlled temperature system. The protein solutions used for the UV measurement were the same utilized for the fluorescence experiments. For the UV spectra of mixtures containing Trp, Tyr, and Met with S or Se in the same ratio as in SsADH solutions were prepared that contained 27, 175, and 122 µM Trp, Tyr and Met, respectively, and 27, 175, 13.5, and 108 µM Trp, Tyr, Met and Se-Met, respectively, in 50 mM glycine-NaOH, pH 9.8. Each spectrum was obtained in duplicate with virtually identical results. Temperature was kept at 25 ( 0.2 °C for all experiments. Special Analysis of Protein 3D Structure. Analysis of characteristic features of tryptophan and tyrosine residues location in 3 D structure of Se-SsADH macromolecule was done on the basis of its atoms coordinates (file Pdb1JVB.ent in the Protein Data Bank).1,2 In this work, a special analysis of the peculiarities of tryptophan and tyrosine residue location in protein that can affect its fluorescence characteristics is used.6-8 In particular, this analysis includes the determination of the conformation of tryptophan and tyrosine residue’s side chain, identification of its nearest neighborhoods along the polypeptide chain and conformation of the part of polypeptide chain where it is located. Special attention is paid to the analysis of the microenvironment of tryptophan and tyrosine residues. Microenvironment of tryptophan and tyrosine residue is determined as a set of atoms that are no greater than r0 distant from the geometrical center of the indole or phenol rings. To take into account all atoms that can contact the indole or phenol ring, the value of r0 was chosen as 7 Å.6-8 For all atoms of microenvironment, the distance from the geometrical center of the indole or phenol ring and their location relative to these rings are determined. The nearest atom of microenvironment to each atom of the indole or phenol ring is specified and the distance between them is determined. One of the characteristics of microenvironment is packing density of atoms that is determined as a number of atoms that compose microenvironment, or the part of microenvironment volume occupied by atoms. The volume of each atom is determined on the basis of its van der Waals radius and only the part of the volume that is inside the microenvironment is taken into account. This estimation is of course not exact because in reality atoms are incorporated by chemical bonds and occupy a smaller volume. Nonetheless, it is not significant for comparative estimation of packing density of microenvironment of different tryptophan and tyrosine residues. Although it is considered that characteristics of tryptophan and tyrosine residue are mainly determined by their close vicinity, the long614
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Figure 1. Fluorescence spectra of SsADH (1) and Se-SsADH (2) solutions of equal optical density (OD280 ) 0.056). Enzyme concentration is 0.043 and 0.015 mg/mL, respectively; buffer 50 mM glycine/NaOH, pH 9.8; temperature 25 °C; λex ) 295 nm.
range effect cannot be excluded. In particular, the efficiency of nonradiative energy transfer from tyrosine to tryptophan residues and between tryptophan and tyrosine residues is evaluated9 W)
1 2/3 R 1+ 2 k R0
()
6
(1)
where R0 is the so-called critical Fo¨rster distance, R is the distance between the geometrical centers of the indole (phenol) rings of donor and acceptor, and k2 is the factor of mutual orientation of donor and acceptor k2 ) (cos θ - 3cosθAcosθD)2
(2)
where θ is the angle between the directions of the emission oscillator of donor and absorption oscillator of acceptor; θA and θD are the angles between the oscillators mentioned above and the vector connecting the geometrical centers of donor and acceptor.10 In this evaluation, R0 is taken from the literature11,12 while other values are determined on the basis of atoms coordinates.6-8 The calculations were done under assumption of rigid oscillators.
Results and Discussion Localization of Tryptophan Residues and Characteristics of Their Microenvironments. A rather blue fluorescence spectrum of SsADH recorded at 295 nm (Figure 1) witnesses that tryptophan residues of this enzyme are located in the low polarity environment and are inaccessible to the solvent. At the same time, analysis of the structure of SsADH monomer shows that tryptophan residues are to a great extent accessible to the solvent (Figure 2). Apparently, the tryptophan residues became inaccessible only after the formation of quaternary structure of native enzyme (tetramer form). Trp95 and Trp117 are located in the Zn containing loop (residues 95-117) of catalytic domain. This loop contains two small β-strands in which tryptophan residues are located (Trp95-Gln96 and Arg116-Trp117) and two R-helixes (helix R2: Cys101-Ile106 and helix R3: Glu108-Cys112). The density of tryptophan residues microenvironment is rather high. Microenvironments of Trp95 and Trp117 consist of 81 atoms, including four atoms of bonded water (d ) 0.77), and 74 atoms, including two atoms of bonded
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Figure 3. Tyrosine contribution in fluorescence spectra of SsADH and Se-SsADH. Curves 1 and 2 are spectra of SsADH and curves 3 and 4 those of Se-SsADH excited at 280 and 295 nm, respectively. All spectra are normalized to unity at 365 nm. Curves 5 and 6 are difference spectra of fluorescence of SsADH and Se-SsADH (1-2 and 3-4), respectively, which characterize the contribution of tyrosine residues.
Figure 2. Tryptophan, tyrosine, and Se atoms of methionine residues localization in Se-SsADH. A. 3D structure of the SsADH subunit apo-form. Tryptophan, tyrosine, and Se atoms of methionine residues are shown as spheres. Se atoms of Met44 occupy two different spatial positions because the side chain of this residue adopts two conformations with 0.6 and 0.4 occupancy. B. Surface of the SsADH subunit apo-form. Trp95 and Trp117 are shown in green and red, respectively. C. Increased surface fragment containing tryptophan residues. This figure is constructed based on Protein Data Bank, file 1JVB.ent,1,2 The drawing was generated by the graphic programs VMD13 and Raster 3D.14
water (d ) 0.71), respectively (Table 1). The side chains of both tryptophan residues have g+ conformation concerning χ1 angle, but belong to different rotamers concerning χ2 (Table 1). A sufficiently large number of polar groups of the amino acid side chains form the microenvironment of Trp95 (Table 2). They are ND1 and NE2 atoms of His68, oxygen atoms OD1 of Asp95 and OG1 of Thr153 and Thr158, as well as sulfur atom SG of Cys154. Furthermore, in the microenvironment of Trp95 there are five complete peptide bonds, four oxygen atoms and one nitrogen atoms of peptide bonds, and four molecules of bonded water (Table 1). In the microenvironment of Trp117 there are only two polar groups belonging to the amino acid side chains: OD1 and ND2 atoms of amide group of asparagine Asn121 (Table 2). At the same time, five complete peptide bonds and three oxygen atoms and two nitrogen atoms of peptide bonds (Table 1), as well as two oxygen atoms of bonded water belong to Trp117 microenvironment (Table 2). Yet, despite many polar groups in the vicinity of Trp95 and Trp117, their microenvironments mainly are formed by hydrophobic
groups of amino acid side chains. Thus, in the vicinity of Trp95 there are CE1 and CZ atoms of Phe49, CG1 and CD1 atoms of Ile153, CB, CG2, and CD1 atoms of Ile157 as well as side chains of Pro94 and Val296. Microenvironment of Trp117 is formed by the side chains of Phe49, Leu58, Pro115, Ile120, Leu295, and Val296. We can conclude that tryptophan residues belong to the hydrophobic cluster which includes Pro94, Pro115 and Phe49, beside Trp95 and Trp117. This location of tryptophan residues can explain a rather blue fluorescence spectrum of SsADH recorded at 295 nm excitation (Figure 1). It is necessary to emphasize that all sulfur atoms of methionine residues are rather far from Trp95 and Trp117. The reasons for the influence of the change of sulfur atoms to selenium ones on the protein fluorescence will be considered later. Microenvironments of Tyrosine Residues. Along with two tryptophan residues, SsADH monomer contains 13 tyrosine residues. When SsADH is excited at 275 nm (in the region of tyrosine maximum contribution in the absorption) and at 280 nm tyrosine residues absorb 63 and 58% from the whole quantity of absorbed quanta (the contribution of tyrosine residues in the bulk absorption of SsADH was calculated on the basis of the data of Mihalyi).15 Usually, to determine the contribution of tyrosine residues to the bulk protein fluorescence the fluorescence spectra excited at 280 and 297 nm are normalized at a red wavelength (e.g., 365 nm), where the fluorescence is determined by tryptophan residues only. If protein fluorescence is determined by tryptophan and tyrosine residues only, these spectra must coincide in the red wavelength region and differ in the blue one if the contribution of tyrosine residues is significant. The comparison of fluorescence spectra excited at 295 and 280 nm shows a significant contribution of tyrosine residues in SsADH fluorescence (Figure 3). The location of individual tyrosine residues in the structure of SsADH macromolecule is such that their properties differ Journal of Proteome Research • Vol. 3, No. 3, 2004 615
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Table 1. Characteristics of Microenvironments and Side Chain Conformation of Tryptophan Residues of Se-SsADHa conformation of the side chain of tryptophan residue and packing density of the microenvironment
aromatic rings and proline residues in the microenvironment
residue
N
d
χ1, (deg)
χ2, (deg)
residue
R (Å)
Trp95
81
0.77
313
111
Phe 49 Pro 94 Trp117
4.4 3.4 3.9
Trp117
74
0.71
291
345
Phe 49 Trp 95 Pro 115
3.6 3.7 3.7
peptide bonds in the microenvironment of tryptophan residue
the whole peptide bond (both O and N atoms enclosed) Pro 94-Trp 95 Trp 95-Gln 96 Trp 117-Leu 118 Thr 153-Cys154 Val 296-Gly 297 Pro 94-Trp 95 Trp 95-Gln 96 His 110-Leu 111 Pro 115- Arg 116 Arg 116-Trp 117 Trp 117-Leu 118
O atoms of peptide bond
N atoms of peptide bond
His 68 Leu 118 Cys 154 Gly 297
Val 296
Gln 96 Glu 109 Cys 112
Pro 115 Asn 121
a N is the number of atoms in the microenvironment of tryptophan residue; d is the density of tryptophan residue microenvironment; χ1 and χ2 are the angles characterizing the conformation of tryptophan residue side chain; R is the minimal distance between aromatic rings or proline residues involved in the microenvironment of tryptophan residue and indole ring.
Table 2. Amino Acid Side Chains-Potential Quenchers of Tryptophan Fluorescence of Se-SsADHa Trp
95
117
residue
atom
N
CA
C
O
CB
CG
CD1
CD2
NE1
CE2
CE3
CZ2
CZ3
CH2
centre
His 68 His 68 Asn 93 Thr 153 Cys 154 Thr 158 HOH HOH HOH HOH Asn 121 Asn 121 HOH HOH
ND1 NE2 OD1 OG1 SG OG1 1 34 188 190 OD1 ND2 9 17
9.43 9.62 2.92 7.90 9.40 9.87
9.94 10.11 3.68 9.07 9.77 9.74
11.36 11.60 4.03 10.23 11.25 10.96
12.33 12.43 4.02 10.77 11.77 10.99
10.03 9.88 3.54 8.87 9.04 8.41
8.80 8.47 3.72 7.64 7.50 7.20 4.83
8.83 8.24 3.32 6.64 6.91 6.56
7.55 7.28 4.96 7.52 6.62 6.88
7.70 6.94 4.40 5.79 5.55 5.80
6.79 6.22 5.26 6.38 5.30 6.02
7.21 7.22 6.08 8.48 7.05 7.56
5.53 4.89 6.57 6.33 4.28 5.90
6.00 6.06 7.25 8.41 6.27 7.44
5.09 4.82 7.43 7.42 4.91 6.65
6.93 6.54 5.31 7.00 5.85 6.44 6.84 6.71 5.61 6.26 5.54 6.38 4.70 4.59
6.23 2.84 4.46
3.50 5.43
3.76 5.91
3.27 5.46
3.45 5.26
4.22 5.57
5.08 6.26 3.51
4.89 5.91
6.08 6.95
6.00 6.77 4.24
5.08 5.97
7.08 7.59
6.29 6.86
3.78 4.99 7.18 7.61 4.24
a
The distance between O, N, and S atoms and all atoms of indole rings of tryptophan residues are shown in Å. For oxygen atoms of bounded water is given the distance only to the nearest atom of indole ring.
significantly (see Table 3). Thus, in the microenvironment of Tyr102 there are only 37 atoms and hence this tyrosine residue can be considered as external, accessible to solvent. The density of the microenvironments of Tyr126, Tyr137, Tyr160, and Tyr267 is significantly higher and these tyrosine residues are internal. In the microenvironments of these tyrosine residues there are 74, 74, 78, and 83 atoms, respectively. The density of microenvironment of oxygen atom of hydroxyl groups of all tyrosine residues (given in brackets) is lower than that of atoms in the sphere with the center in the geometrical center of the phenol ring. This means that hydroxyl groups of the majority (or all) tyrosine residues are directed from inside to the macromolecule periphery. This is fairly common for tyrosine residues located near the surface of the macromolecule (residues with low density of microenvironment) Tyr102, Tyr103, and Tyr219 and much less typical for internal Tyr267, Tyr126, and Tyr137. As is well-known,16,17 the proton-acceptor groups located in the close vicinity of tyrosine residues have the greatest influence on their fluorescence properties. So Tyr126 and Tyr267 are likely quenched by carboxyl groups of asparagine acids Asp41 and Asp246, respectively. Some atoms of the backbone can also be regarded as potential quenchers: oxygen atoms of Tyr135 and Met138 for Tyr84 (R ) 3.75 and 3.48 Å, respectively), oxygen atom of Glu80 for Tyr135 (3.52 Å), oxygen atom of Gly97 for Tyr137 (3.51 Å), and oxygen atom of Thr253 for Tyr267 (3.72 Å). There are 616
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selenium atoms of methionine residues in the microenvironment of several tyrosine residues: selenium atoms of Met138, Met1, Met306, and Met269 in the microenvironments of Tyr84, Tyr129, Tyr139 and Tyr267, respectively. At the same time, in all of these cases, sulfur (selenium) atoms are far from the center of the phenol ring of the tyrosine residue and its hydroxyl group. That is why the direct quenching of sulfur (selenium) atoms on the fluorescence of tyrosine residues is not evident. It is possible that the change of sulfur atom for selenium may influence the fluorescence of Tyr84 through the change of the distance between the hydroxyl group of this tyrosine residue and oxygen atoms of backbone Tyr135 and Met138, or through the change of quenching properties of oxygen atoms of these groups. Tyrosine residues can significantly affect tryptophan fluorescence due to nonradiative energy transfer (sensitization of tryptophan fluorescence). Nonradiative Energy Transfer Trp T Trp, Tyr f Trp, and Tyr T Tyr. Analysis of the localization of tryptophan and tyrosine residues in the 3D structure of protein macromolecule allowed us to evaluate the efficiencies of tryptophan-tryptophan, tyrosine-tryptophan, and tyrosine-tyrosine energy transfer (Tables 4-6). The distance between the geometrical centers of the indole ring of tryptophan residues is only 5.76Å. Therefore, even though the planes of their indole rings are practically perpendicular to each other (orientation factor is very small k2 ) 0.04), the efficiency of energy transfer between
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Highly UV-Absorbing Complex in Se-SsADH Table 3. Characteristics of the Microenvironments of Tyrosine Residues of Se-SsADH
residue
Na
Tyr 84
66 (56)
Tyr 102 Tyr 103
37 (26) 50 (27)
Tyr 126
74 (72)
Tyr 129
57 (38)
Tyr 135
62 (45)
Tyr 137
74 (61)
Tyr 139
60 (46)
Tyr 160
Oxygen atoms of bounded water in microenvironment of tyrosine residues
amino acid side chains potential quenchers of tyrosine fluorescence
RC (Å)c
OD2 Asp 88 SE Mse 138 NZ Lys140
5.8 5.3 5.3
ROH (Å)d
7.8 6.0 6.4
Oxygen atoms of backbone in microenvironment of tyrosine residues
NHOHb
RC (Å)c
ROH (Å)d
residues
RO-OH (Å)e
60 142 167
5.2 7.0 6.8
4.0 >8.0 >8.0
Val 82 Gly 83 Tyr 135 Lys 136 Mse 138 Tyr 137 Tyr 139
5.87 5.32 3.75 5.04 3.48 6.79 6.45
67 176 67 176
4.5 6.6 5.4 6.5
3.0 >8.0 5.4 7.0
Leu 111
6.19
10 18 19
5.5 4.9 5.2
5.1 4.1 5.7
Ala 35 Gly 36 Val 37 Cys 38 Asp 41 Leu 66 Ala 327 Glu 128
6.17 4.46 6.31 5.90 5.90 6.04 5.58 5.38
Pro 26 Glu 80 Val 81 Val 82 Asn 93 Trp 95 Glu 96 Gly 97 Glu 98 Glu 109
6.73 3.52 5.88 4.90 6.85 5.26 5.29 3.51 6.82 6.25
Lys 140 Leu 303 Mse 306 Arg 307
5.46 5.05 4.82 5.70
Ser 300 Asp 301 Leu 303 Gly 304
4.74 5.12 6.15 4.65
Asp 218 Ile 235
5.52 5.76
Pro 228 Lys 252 Thr 253 Val 202 Asp 203 Ile 221 Ala 223 Val 244 Ile 245 Asp 426 Thr 253 Leu 254 Val 256 Tyr 257 Val 268 Pro 256 Lys 259 Leu 261 His 280 Leu 283
6.67 6.86 6.80 6.08 6.20 6.88 6.37 6.54 6.44 6.32 3.72 5.60 6.63 5.20 6.36 4.16 6.37 6.64 6.08 5.34
SG Cys 101 OD1 Asp 113 OD2 Asp 113 OD1 Asp 41 OD2 Asp 41
6.1 4.9 6.1 5.4 4.7
>8.0 5.9 7.0 3.9 2.8
SE Mse 1 NZ Lys31 OE1 Glu 128 OE2 Glu 128 ND1 His 134 NE2 His 134
5.5 5.6 5.5 6.7 4.2 4.5
7.3 3.9 4.7 4.6 5.4 4.8
16 89
5.4 4.6
5.8 2.8
65
6.1
5.2
OD1 Asn 93 ND2 Asn 93 OE1 Gln 96 NE2 Gln 96 ND1 His 134 NZ Lys 136 OE1 Gln 299 NE2 Gln 299 SE Mse 306 OE1 Glu 310 OE2 Glu 310
6.2 5.3 4.1 4.3 6.2 5.2 4.9 4.6 5.9 6.4 6.9
7.5 6.5 5.1 6.1 7.0 4.2 6.3 5.7 7.5 6.3 7.3
1 11 21 80 94
6.0 7.0 4.4 4.7 6.0
6.1 6.2 4.2 3.0 3.7
78 (54)
NE Arg 164 OD1 Asp 301 OD2 Asp 301
5.5 4.9 6.7
5.7 5.6 7.7
Tyr 219
51 (33)
OD1 Asp 218 OD2 Asp 218 NH1 Arg 234 NH2 Arg 234
6.8 6.7 6.7 6.6
7.4 7.3 7.0 7.6
44 56 57 60 84 51 71 110 119 158 122 124 140 141 146 155 162 192 193
6.5 5.3 4.4 4.4 4.3 3.3 5.2 4.3 5.2 6.1 5.4 6.1 6.4 4.3 6.8 5.3 5.4 5.5 4.3
4.6 4.5 4.3 5.6 2.6 4.4 4.6 2.7 5.2 5.0 5.9 8.0 6.2 2.7 7.0 4.2 4.9 4.3 2.8
Tyr 257
62 (58)
OG1 Thr 253
6.7
8.0
Tyr 267
83 (82)
OD1 Asp 246 OD2 Asp 246 ND2 Asn 249 OG1 Thr 253 SE Mse 269
6.0 4.3 5.3 6.5 5.8
4.4 2.5 4.9 4.4 7.0
Tyr 279
60 (55)
OG Ser 287
6.8
5.5
69
4.0
5.1
a N is the number of atoms in the microenvironment of tyrosine residue. b N c HOH is the number of the molecule of bounded water. RC is distance from the geometrical center of tyrosine residue. d ROH is distance from the oxygen atom of hydroxyl group of tyrosine residue. e RO-OH is distance between oxygen atom of the backbone and hydroxyl group of tyrosine residue.
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Table 4. Nonradiative Energy Transfer between Tryptophan Residues Trp 95 and Trp 117 of Se-SsADH R (Å)a
k2c
R0 (Å)b
Wd
5.76
0.04
0.64-0.77
0.28-0.43
a R is the distances between the geometrical centers of the indole rings of tryptophan residues; b R0 is Forster radius for tryptophan residues;11,12 c 2 k is orientation factor; d W is the value of the efficiency of nonradiative energy transfer.
Table 5. Efficiency of Nonradiative Energy Transfer from Tyrosine to Tryptophan Residues of Se-SsADH Tyr/Trp
Trp 95
Trp 117
Tyr 84 Tyr 102 Tyr 103 Tyr 126 Tyr 129 Tyr 135 Tyr 137 Tyr 139 Tyr 160 Tyr 219 Tyr 257 Tyr 267 Tyr 279
0.68 0.50 0.17 0.10 0.14 0.24 0.96 0.83 0.95 0.02 0.00 0.01 0.10
0.49 0.64 0.98 0.79 0.05 0.25 0.97 0.38 0.17 0.03 0.00 0.21 0.09
Trp95 and Trp117 is rather high (W ) 0.43, Table 4). Table 5 shows that there is a rather high efficiency of energy transfer from seven tyrosine residues (Tyr84, Tyr102, Tyr103, Tyr126, Tyr137, Tyr139, Tyr160) to tryptophan residues. Thus, the contribution of these tyrosine residues to the bulk SsADH fluorescence is negligible. Although the efficiency of energy transfer from Tyr129 and Tyr135 to tryptophan ones is small, they also can be regarded as quenched. The efficiency of nonradiative energy from Tyr135 to Trp95 and Trp117 is 0.24 and 0.25, respectively. At the same time, the efficiency of energy transfer between Tyr135 and Tyr137 is rather high (W ) 0.86, Table 6), but as Tyr137 transfers its excitation energy to Trp95 the energy transfer is directed from Tyr135 to Tyr137:
The efficiency of nonradiative energy transfer between Tyr129 and Tyr126 is lower than that between Tyr135 and Tyr137. Nonetheless, Tyr129 also can be regarded as quenched, because in this case there is directed energy transfer from Tyr129 through Tyr126 to Trp117:
Consequently, only four tyrosine residues (Tyr219, Tyr257, Tyr267, and Tyr279) which are located in coenzyme binding domain, can give the noticeable contribution to SsADH fluorescence. Tyr84, Tyr102, Tyr103, Tyr126, Tyr129, Tyr135, Tyr137, and Tyr139 which are located in the catalytic domain and Tyr160 which is located between the catalytic and coenzyme binding domains do not contribute significantly in the bulk fluorescence of enzyme due to effective nonradiative energy transfer to tryptophan residues. 618
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Influence of Sulfur-Selenium Substitution to Fluorescence of SsADH. The change from the sulfur to selenium atom of methionine in SsADH leads to the decrease of intrinsic fluorescence of the enzyme by 40% (ref 4; Figure 1). At the same time, it appears that all sulfur atoms of methionine residues which are replaced by selenium are located sufficiently far from both Trp95 and Trp117. The nearest sulfur atom of methionine Met44 is situated at 10.5 and 9.2 Å from the geometrical centers of indole rings of Trp95 and Trp117, respectively. Thus, the decrease of the fluorescence intensity of SsADH when Met is substituted by Se-Met can be caused by the following: (1) change of the macromolecule structure, although selenomethionine-substituted protein exhibited enzymatic and dichroic activity similar to that of the wild-type protein;1 (2) existence of some long-range quenching of tryptophan fluorescence by selenium, although no mechanism is known yet; or (3) indirect influence of Se on tryptophan fluorescence via other groups of atoms. In particular, it must be mentioned that Se atoms of Met138 and Met306 are located in the vicinity of Tyr84 and Tyr139, respectively (5.3 and 5.9 Å from the center of phenol rings of these residues) which transfer their energy of excitation to tryptophan residues (Table 5). The quenching of Tyr84 and Tyr139 when sulfur atoms are changed by selenium ones in the Met138 and Met306 could explain the diminishing SsADH fluorescence excited at 280 nm by diminishing of energy transfer from these tyrosine residues to tryptophan. It is necessary to mention that the fluorescence spectra shown in Figure 1 were recorded for SsADH and Se-SsADH solutions of the same optical density. At the same time, we noted that these solutions have significantly different concentration. Absorption spectra of SsADH and Se-SsADH solutions of the same concentration are presented in Figure 4 (curves 1 and 3). Absorption spectra with the deduction of the apparent optical density determined by light scattering (curves 5 and 6) are shown in Figure 4 (curves 2 and 4) and Figure 5. The results suggest that the substitution of sulfur by selenium in methionine residues of SsADH leads to the significant change in absorption spectrum. It is known that sulfur-containing amino acids (cysteine and methionine) do not contribute significantly to UV absorption spectra of proteins.18 The comparison of absorption spectra of methionine and Se-methionine (Figure 5, Insert A), and the equivalent mixture of tryptophan, tyrosine, methionine, and Se-methionine residues (Figure 5, Insert B) suggests that although Se-methionine has a more intensive absorption band in comparison with that of methionine, the contribution of Se-methionine in the bulk absorption spectrum of the equivalent mixture is negligible. Consequently, a significant change in absorption spectrum of SsADH when sulfur is substituted by Se can be explained only by the formation of intensively absorbing complex between Se and some neighboring group, presumably nearby tyrosine residues. The possible complexes are SeMet138 and Tyr84; SeMet1 and Tyr129; SeMet306 and Tyr139; SeMet269 and Tyr267. This result is important not only for SsADH. Selenomethionine incorporation is a procedure commonly used to assist in solving the crystallographic 3D structure of macromolecules; thus, similar formation of complexes can also occur in other proteins when S is substituted by Se. It is evident that to compare fluorescence characteristics of SsADH and Se-SsADH it is necessary to take solutions of equal concentration (Figure 6), but not of equal optical density (Figure 1) as was done previously.4 Tryptophan fluorescence intensity of the SsADH and Se-SsADH solutions of equal concentration is practically the same (Figure 6).
research articles
Highly UV-Absorbing Complex in Se-SsADH Table 6. Efficiency of Nonradiative Energy Transfer between Tyrosine Residues of Se-SsADHa Tyr 84
Tyr 84 Tyr 102 Tyr 103 Tyr 126 Tyr 129 Tyr 135 Tyr 137 Tyr 139 Tyr 160 Tyr 219 Tyr 257 Tyr 267 Tyr 279
0.00 0.02 0.02 0.00 0.00 0.65 0.81 0.02 0.00 0.00 0.00 0.00
Tyr 102
Tyr 103
Tyr 126
Tyr 129
Tyr 135
Tyr 137
Tyr 139
Tyr 160
Tyr 219
Tyr 257
Tyr 267
Tyr 279
27.96
26.60 4.84
23.60 32.64 28.28
17.55 37.55 34.42 15.30
6.42 24.81 23.96 25.52 19.44
11.29 17.97 15.83 20.03 20.44 9.58
9.28 26.67 25.04 23.14 22.41 14.06 12.90
19.91 21.53 19.68 24.95 30.55 22.41 16.24 11.92
42.43 39.86 37.98 39.76 49.56 45.97 39.48 33.36 23.82
41.52 33.35 30.31 32.23 45.22 43.70 35.12 34.03 23.54 15.70
38.04 25.93 23.15 31.01 43.23 39.23 30.30 31.22 20.08 19.58 7.85
45.39 28.35 26.30 38.25 50.77 45.83 36.73 39.00 27.62 24.60 13.09 8.70
0.94 0.00 0.00 0.00 0.12 0.01 0.01 0.00 0.00 0.02 0.00
0.00 0.01 0.00 0.44 0.01 0.00 0.00 0.00 0.01 0.01
0.28 0.01 0.01 0.00 0.02 0.00 0.00 0.01 0.00
0.10 0.05 0.04 0.00 0.00 0.00 0.00 0.00
0.86 0.46 0.01 0.00 0.00 0.00 0.00
0.68 0.01 0.00 0.00 0.00 0.00
0.52 0.00 0.00 0.00 0.00
0.03 0.00 0.00 0.00
0.13 0.06 0.00
0.97 0.21
0.28
a
The table shows the values of the efficiency of nonradiative energy transfer W (lower left part of the table) and the distances between the geometrical centers of the phenole rings of tyrosine residues (upper right part of the table).
Figure 4. Absorption spectra of SsADH and Se-SsADH. Curves 1 and 3 are absorption spectra of solutions of Se-SsADH and SsADH, respectively, having equal concentration 0.105 mg/mL. Curves 2 and 4 are absorption spectra of solutions of Se-SsADH and SsADH, respectively, with the deduction of apparent optical density Dscat ) aλ-n, determined by light scattering (1-5 and 3-6, respectively). The insert shows supplementary curves which elucidate the procedure of subtraction of the scattered light in the absorption spectrum of Se-SsADH. Curve 1 is the dependence of ln(D) from ln(λ), curve 2 is a straight line, extrapolating the linear region of the curve ln(D) ) f(ln(λ)), corresponding to the spectral region where active absorption is absent; ln(Dpacc) ) a - n ln(λ), a ) 7.22, n ) 3.44.
Nonetheless, the spectrum position of Se-SsADH is somewhat more red in comparison with that of SsADH (Figure 6). The reasons for the difference obtained between tryptophan residue characteristics of SsADH and Se-SsADH cannot be explained from the analysis of the 3D structure of Se-SsADH. It is possible that these differences can be due to some differences of 3D structure of SsADH and Se-SsADH, and/or due to the greater content of aggregates in the solution of Se-SsADH, also indicated by the difference in light scattering of the solutions of SsADH and Se-SsADH (see Figure 4). The fluorescence excitation spectra of SsADH and Se-SsADH recorded at 320 and 365 coincide. This means that methionine residues with sulfur atoms replaced by selenium do not participate in fluorescence and do not transfer excitation energy to tryptophan or tyrosine residues. Abbreviations used: SsADH, alcohol dehydrogenase from Sulfolobus solfataricus; Se-SsADH, selenomethionine-substituted SsADH.
Figure 5. Effect of substitution of the sulfur for selenium atom of methionine residues on the absorption of SsADH. (1) absorption spectrum of Se-SsADH, (2) absorption spectrum of SsADH and (3) difference spectrum. Insert A. Absorption spectra of Met (curve 2) and Se-Met (curve 1) solutions. Concentration (108 µM) is equivalent to that of Met residues in protein solution of 0.105 mg/mL. Insert B. Absorption spectra of equivalent mixtures of Trp, Tyr, Met, and SeMet taken in the ratio 2:13:1:8 (curve 1) and in the ratio 2:13:9:0 (curve 2); curve 3, difference spectrum.
Figure 6. Fluorescence spectra of SsADH (1) and Se-SsADH (2), λex ) 295 nm. Protein concentration is 0.043 mg/mL.
Acknowledgment. This work was supported in part by grants from INTAS (2001-2347) and from Presidium of Russian Academy of Sciences for the program “Physicochemical Biology”. Journal of Proteome Research • Vol. 3, No. 3, 2004 619
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