Domain structural flexibility in rhodanese examined by quenching of a phosphorescent probe

236

Biochimica et Biophysica Acta 916 (1987) 236-244

Elsevier BBA 32980

D o m a i n s t r u c t u r a l f l e x i b i l i t y in r h o d a n e s e e x a m i n e d by q u e n c h i n g of a phosphorescent Henryk

Koloczek

probe

a and Jane M. Vanderkooi

b

Institute of Molecular Biology, Jagiellonian University, Cracow (Poland) and h Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, PA (U.S.A.

(Received 23 June 1987)

Key words: Rhodanese domain structure; Domain structure; Eosin; Phosphorescence; Protein tertiary structure

Rhodanese (thiosulfate: cyanide suifurtransferase, EC 2.8.1.1) is an enzyme composed of two domains with the catalytic site located in the bottom of the crevice formed by the two domains. In this work, rhodanese was labeled at its catalytic site with the phosphorescence probe eosin isothiocyanide. The accessibility of molecules to the probe was determined by phosphorescence lifetime-quenching studies. The phosphorescent probe was much more accessible to small molecules (I - and thiosulfate, radius about 3 - 5 A) than to a larger molecule (spin-label probe TEMPO, radius about 8 - 1 0 ~,). It was observed that a temperature-induced change in the rate of quenching occurred at around 28 ° C. The results are interpreted in terms of structural fluctuations and displacement in the domains.

Introduction Rhodanese (thiosulfate : cyanide sulfurtransferase, EC 2.8.1.1) is a w i d e l y d i s t r i b u t e d mitochondrial enzyme whose physiological role is not fully known, but appears to be involved in the removal of toxic cyanide and sulfide, and it is perhaps also involved in the synthesis of proteinb o u n d n o n - h e m e iron-sulfur centers [1,2]. The most studied rhodanese-catalyzed reaction in vitro is the transfer of the outer sulfur of thiosulfate ($20 3) to the nucleophilic acceptor, C N - , by a double

Abbreviations: Rh, rhodanese; EITC, eosin isothiocyanide; RhSS, Rh with the transferable sulfur of thiosulfate bound at the active site in a persulfide bond; RhEITC, Rh with EITC at the active site; Rh(SS)EITC, RhSS with EITC labeled not at the active site; TEMPO, 2,2,6,6-tetramethyl-l-piperidinyloxy. Correspondence: J.M. Vanderkooi, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.

displacement reaction. During the course of this reaction, the enzyme cycles through two stable catalytic intermediates: the sulfur-substituted (RhSS) and the free form (Rh). A n u m b e r of studies have shown that rhodanese in solution has structural flexibility and that a reversible structural change accompanies catalysis [3-6]. X-ray studies of the rhodanese have demonstrated that its single polypeptide chain of 293 amino-acid residues is folded into two nearly equal size domains which are connected by a single polypetide chain tether [7]. The domains fold to form a structure resembling a clam, the active site being in the cleft formed by the juxtaposition of the two domains. The sulfur atom transferred during catalysis is b o u n d in a persulfide linkage to SH of cysteine-247 at the b o t t o m of the cleft. The persulfide is stabilized by hydrogen bonds between the sulfur atom and N H and O H groups of the b a c k b o n e polypeptide chain [7-9]. In addition to a conformational change due to the substrate, the sulfur-free (Rh) and the sulfur-

0167-4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

237 substituted (RhSS) enzymes appear to undergo a temperature-related structural transition at 27 and 29°C, respectively, as indicated by tryptophan fluorescence intensity and extrinsic dyes' fluorescence polarization [10] and by nuclear magnetic resonance [11]. Based upon these studies, it was suggested that the thermal transition observed in rhodanese results from changes in the interaction between the two domains. In this paper, we examine the structural flexibility of rhodanese using a phosphorescence technique which allows us to study the accessibility of various small molecules to the phosphorescent label [12,13]. For labeling of the enzyme's active pocket, eosin isothiocyanide (EITC) was used. The presence of the four bromine atoms in the structure promotes intersystem crossing from the lowest excited singlet state to the triplet state with a quantum efficiency about 70% in aqueous solutions [14]. This label was covalently attached to the sulfur which is at the active site at the bottom of the crevice. Quenching of phosphorescence allows a measurement of the penetration of small quencher ions and molecules to the active pocket. We also examined the phosphorescence quenching of a probe which labeled Rh under conditions that the active site was blocked, i.e., the label is not at the active site. We see variations in accessibility of small molecules to the phosphorescence probe as a function of quencher size and as a function of temperature. We interpret our results in terms of thermal induced conformational changes and the segmental flexibility of domains. Materials and Methods

Bovine liver rhodanese was prepared according to the procedure of Horowitz and DeToma [15] and was stored as a microcrystalline suspension at 4 ° C in 3.25 M (NH4)2SO 4 in the presence of 1 mM Na2S203. The protein was homogeneous according to sodium dodecyl sulfate-acrylamide gel electrophoresis. The protein concentration was determined using an absorption coefficient at 280 nm of 1.75 for a 0.1% solution [16]. The molecular weight was assumed to be 33 000 [8]. Eosin isothiocyanide (EITC) was purchased from Molecular probes (Eugene, OR). Tetramethyl-l-piperidinyloxy (TEMPO) and methyl-

vinylketone were obtained from Aldrich Chemicals (Milwaukee, WI). Sigma Chemicals (St. Louis, MO) supplied the enzymes used for removal of oxygen (glucose oxidase and catalase).

Labeling of rhodanese by EITC Typically, the conjugation of EITC was carried out in the following manner. The stock solution of rhodanese was passed over a G-25 Sephadex column to remove ammonium sulfate and excess thiosulfate. An EITC solution in 0.01 M Tris-HCl buffer (pH 8.0) was added to 1 ml of 10 ktM rhodanese in the Tris-HC1 buffer containing 50 ~M KCN to give a 5-fold molar ratio of E I T C / protein. (The presence of KCN ensures that the protein is predominantely in the Rh form.) The second way of labeling rhodanese was carried out in same manner, but KCN was not included in the reaction mixture; under these conditions the SH group of Cys-247 remains blocked by thiosuifate [17]. Aliquots of the reaction mixture were taken and the activity of rhodanese was measured by the colorimetric method of Sorbo [16]. The time-course of inactivation of both forms of rhodanese and the E I T C / p r o t e i n radio are shown in Fig. 1. The labeled protein was then separated from unreacted dye on a G-25 column equilibrated with 0.01 M Tris-HC1 buffer (pH 8.0) at 4°C. All manipulations of the enzyme were done in the dark. The dye concentration was determined using an absorption coefficient of 8.3.104 M ~-cm -1 at 528 nm [18]. Because the dye absorbs at 280 nm, it was necessary to take this into consideration when determining the enzyme concentration by its absorbance at 280 nm (see above). The absorption coefficient for eosin at 280 nm was taken to be 2.15-104 M - l . c m -1 The enzyme was used within 1-2 days after labeling.

Fluorescence and phosphorescence measurement Fluorescence and phosphorescence spectra were obtained on a Perkin-Elmer LS-5 luminescence spectrophotometer. Fluorescence lifetime measurements used an excitation wavelength of 305 nm from a modelocked argon laser pumping a cavity-dumped tunable dye laser. An emission monochromator was used to avoid Raman and other scattering artifacts. A polarizer was inserted at the magic

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Fig. 1. Time-course of inactivation and EITC binding by rhodane,~ (Rh) and sulfur-substituted rhodanese (RhSS). Phedaoes= (20 FM) was incubated at 22°C with a 5-fold molar excess of t_ rC in 0.01 M Tris-HCl buffer (pH 8.0) in the absence or pi,.~.'nce of 50 /.tM KCN. After the indicated time, small amounts of sample were taken and excess unbound EITC was separated on a Sephadex G-25 column at 4°C as described in Materials and Methods: activity relative to unreacted enzyme incubated with KCN (11) or without KCN (e), molar ratio EITC/Rh for reacted enzyme in the presence (A) or absence ( + ) of KCN. angle before the photomultiplier to avoid polarization artifacts on the decay curve. Fluorescence decay (547 nm) was followed by p h o t o n counting. The data were fit to a single-, double- or triple-exponential decay function with lamp deconvolution. Phosphorescence lifetime measurements were p e r f o r m e d with a lab-built instrument described elsewhere [19]. D e c a y was fit by c o m p u t e r to a single- or double-exponential function. For phosphorescence measurements, it is necessary to remove oxygen. To achieve this, an oxygen-consuming enzyme system was included in the sample. This system consisted of glucose oxidase (25 ~ g / m l ) , catalase (8 k t g / m l ) and 0.4% glucose. The samples were covered with a layer of mineral oil and the cuvette was capped.

Analysis of data The apparent rate constant for collisional quenching (kq) was obtained from measurements of decay of phosphorescence according to the Stern-Volmer equation [20]: = 1 + %kq[Ql,

Results

Emission characteristics of EITC, RhEITC and Rh(SS)EITC Time,

%/'r

where % and ~" are the phosphorescence lifetimes in the absence and presence of a given quencher concentration. The energy of quenching activation, E a, was calculated by the Arrhenius equation: kq = A exp( - E J R T).

Fig. 2 compares the excitation, p r o m p t fluorescence, and phosphorescence spectra of EITC, R h E I T C and Rh(SS)EITC complexes in 0.01 M Tris-HC1 ( p H 8.0) buffer. In Fig. 2A the excitation spectra are shown; the excitation maxima are found at 520, 541 and 523 n m for EITC, R h E I T C and Rh(SS)EITC, respectively. The R h E I T C and Rh(SS)EITC excitation spectra show an additional shoulder at 280 nm, which is absent in EITC. Since tryptophan absorbs in this region, the extra b a n d is likely to be due to energy transfer from t r y p t o p h a n to EITC. This shoulder is relatively smaller in the Rh(SS)EITC case. The p r o m p t fluorescence m a x i m u m emission occurs at 539 and 547 n m for E I T C and R h E I T C complex, respectively (Fig. 2B). The emission m a x i m u m for R h ( S S ) E I T C is at 546 n m (data not shown). These values c o m p a r e with emission maxima for E I T C of 541, 543 and 546 n m in buffer, methanol and ethanol, respectively. The room-temperature phosphorescence emission-maximum occurs at 7 8 1 , 6 8 7 and 684 nm for EITC, R h E I T C and Rh(SS)EITC, respectively (Fig. 2C). All complexes also show delayed fluorescence which have the same emission spectra as p r o m p t fluorescence and has the same lifetime as phosphorescence. The phosphorescence excitation spectra of these derivatives are identical to the fluorescence excitation spectra, showing that phosphorescence originates from the same absorption process as fluorescence. The fluorescence decay profiles of E I T C and R h E I T C are shown in Fig. 3. The fluorescence of R h E I T C is non-single exponential (Fig. 3A) with a short c o m p o n e n t of 0.5 ns and a longer c o m p o nent of 2.1 ns, when the fit was constrained to two-exponential functions. Better fit was achieved using three-exponential functions; however, the

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small contribution of a shorter lifetime component ( 0 . 3 2 n s ) ( F i g . 3B).

Phosphorescence decay of eosin, E I T C and R h E I T C data are not precise enough to distinguish this case from the case of a distribution of lifetimes. F r e e E I T C h a s a l i f e t i m e o f a b o u t 1.1 ns, but h a s a

T h e p h o s p h o r e s c e n c e d e c a y c u r v e s o f free E I T C a n d R h E I T C a r e s h o w n in Fig. 4. T h e p h o s p h o r e s c e n c e l i f e t i m e s a t 103C a r e 0 . 7 4 m s a n d

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Fhe temperature dependence of the fluorescence and phosphorescence intensity of RhEITC is shown in Fig. 5. The fluorescence intensity changes little in the temperature range of 10 to 35 o C. The rate constant of phosphorescence decay (reciprocal lifetime) of RhEITC decreases by about 30% in this range; the lifetime of EITC changes by about 20%. Over this temperature range, no changes in the excitation or emission spectra were detected. In the concentration range of 1 to 10 #M, the phosphorescence lifetime of RhEITC was independent of concentration. The lifetime of free ETIC decreases as the concentration increases due to self-quenching [19]; extrapolation to zero EITC concentration, using the Stern-Volmer relationship, gives a lifetime for EITC in buffer of 0.8 ms. We conclude, therefore, that E I T C in the protein has a phosphorescence lifetime less than in aqueous solution.

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A series of molecules were examined for their effect on the phosphorescence lifetimes of eosin and its derivatives. In the absence and presence of the quenching molecule, the decay curves could be fit by single-exponential functions. Typical SternVolmer plots are shown in Fig. 6, illustrating that simple linear relationships between inverse lifetime and quencher concentration of methylvinylketone were obtained for EITC and RhEITC at a range of temperatures.

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241 8"

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Fig. 6. The EITC (open symbols) and RhEITC (closed symbols) phosphorescence quenching curves by methylvinylketone in 10 ° C (zx,i), 22 ° C ([3,1), and 36 ° C (C),l). The protein and dye concentrations are given in Fig. 2C. Excitation and emission wavelength were 517 and 686 nm, respectively.

A summary of the Stern-Volmer quenching constants for eosin and EITC is given in Table I. In the concentration range in which the phosphorescence was quenched, the fluorescence intensity was not affected by any of these molecules. N o difference in quenching efficiency was observed between eosin and EITC. The quenching constants decreased by about a factor of 80-100 when 85% glycerol was substituted for water in the buffer. This change increases the viscosity from about 1 to 100 cP [21].

of phosphorescence

It was noted in the Stern-Volmer plots presented in Fig. 6 that the slopes for EITC phosphorescence quenching are changing gradually between 10, 22 and 35°C, but for the RhEITC complex the slope at 36 ° C is remarkably different from the others (10 and 22 ° C). This phenomenon was studied in detail for a series of quenchers. The temperature dependence of the second-order rate constant is shown in Fig. 7 in an Arrhenius representation for RhEITC, EITC and Rh(SS)EITC. The highest position curves represent the quenching of EITC; in all cases, the dependency upon temperature was continuous. The dependency of log kq vs. T-1 for RhEITC quenched by TEMPO, methylvinylketone or SzO3 shows a discontinuity at about 26 ° C. This discontinuity was reversible. For the smallest quencher, KI, a break in the curves was not detected. This break was also not observed for the phosphorescence-quenching profile of Rh(SS)EITC by T E M P O (Fig. 7a), or by methylvinylketone, thiosulfate or KI (data not shown). The energy of activation, E a, for the quenching constant was 3 to 5 k c a l / m o l for EITC (Fig. 7, legend). At temperatures above and below the transition temperature, the energy at activation for the quenching rates of RhEITC phosphorescence showed similar low energies of activation.

Discussion

Phosphorescence to study domain motion in proteins TABLE I PHOSPHORESCENCE QUENCHING OF EOSIN AND EITC n.t., not tested; buffer, 0.01 M Tris (pH 8.0); temperature, 22o C. (Kq, l/mol per s). Quencher

Eosin (buffer)

Eosin (85% glycerol)

E1TC (buffer)

TEMPO

1.1-109

3 • 107

1 • 109

Methylvinylketone KI KBr KCI KCN Thiosulfate

1 •107 1.2.10s 1.2.10 7 4 •105 6 •103 n.t. no effect up to 0.5 M 1.7. l0 s n.t. 3.8.10 s n.t.

9.1 •106 1.4.107 n.t. 1.4- l0 s 3.9-105

A structural feature of many proteins is their organization into domains, where the domain is a relatively rigid structure which is connected to another domain by a flexible hinge [22]. Structural analogies between domains from proteins of diverse functions are widespread; for example, the adenine- and nucleotide-binding proteins, lactic dehydrogenase, lactate dehydrogenase (soluble), liver alcohol dehydrogenase and glyceraldehyde phosphate dehydrogenase all show similar domain arrangement. Interestingly, rhodanese, flavodoxin, phosphoglycerate kinase, substilisin and adenylate kinase also show striking similarity to the dehydrogenases, even though their functions are not similar. (A cartoon representation of these pro-

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teins is given by Cantor and Schimmel [23].) Rhodanese is the simplest of the domain proteins in that it is a monomer and is composed of only two domains. Rhodanese has a free sulfhydryl at the active site, which enables us to label the crevice region with an optical probe. Based upon hydrodynamic considerations, if the domains show structural flexibility, the time-scale of these motions should be on a time-scale of microseconds or longer. A means to study these slow motions in domain proteins is to use phosphorescence, since penetration of quencher molecules to intrinsic or extrinsic phosphorescence probes would reveal motion on the slow time-scale. (Phosphorescence polarization could be used to measure slow motions [24]; but for soluble proteins, the rotation of the whole protein would cause depolarization, so information on segmental motion is lost.) A series of small molecules and ions were examined for their effect on the phosphorescence of eosin (Table I). Both substrates quench eosin phosphorescence, although the efficiency of quenching is less than 100%. Of the halides, only the quenching of iodide is significant. The quenching constant for the spin-label probe, TEMPO, approaches that for a diffusion-limited reaction. The fluorescence of eosin located in the pocket of rhodanese resembles eosin in an hydrophobic environment. The fluorescence emission maximum is at 540 and at 547 nm for the free- and activesite-bound forms, respectively. However, unlike eosin in a solvent, the decay of the enzyme-bound form is non-single exponential. The phosphorescence emission maximum is at 681 nm and at 687 nm, respectively. The form labeled at a non-active site (Rh(SS)EITC) has an emission maximum intermediate between the two. Energy transfer occurs from tryptophan to eosin, as can be seen in the excitation spectra of RhEITC and Rh(SS) EITC; the transfer was more for the derivative in which eosin is in the active pocket. From the X-ray analysis, it is known that two tryptophan moieties line the active pocket [7,8]; close proximity of the eosin to the tryptophans can account for the observed efficient energy transfer. The decay of phosphorescence was fit to a single exponential within the errors of measurement (Fig. 4) and the Stern-Volmer plots of phosphorescence quenching were linear (Fig. 6).

243 Interdomain flexibility has been observed in many proteins, including hexokinase and immunoglobulins, to name a few [22,25]. Taking as a model that the domains of rhodanese likewise show flexibility, the data presented in this paper can be interpreted in terms of the kinetics of the dynamic equilibration between open and closed forms. If the domains show flexibility, then during the lifetime of the excited singlet state (1-2 ns), decay from eosin which is more or less exposed to the solvent is possible and non-single exponential decay is predicted, as was observed. The lifetime of phosphorescence is much longer: 0.5 to 1 ms. On this time-scale, there appears to be one species. This result can be obtained if there are fluctuations which are rapid on this time-scale. Hence, we can bracket the time-scale of the fluctuations between ns and ms, and they must be in the #s time regime. Addition of quenchers produces quenching of phosphorescence of EITC and RhEITC which follow simple bimolecular kinetics as is evident in the linear Stern-Volmer plots (Fig. 6) and the exponential decay behavior in the presence of quenchers. Again, this suggests that structural fluctuations must occur on the time-scale of /~s. The protein turnover time is much slower than this, about 330 molecules of thiocyanate formed from thiosulfate and cyanide per second per molecule enzyme at 2 0 ° C [16]. The turnover time is therefore not limited by fluctuations. A discontinuity in Arrhenius plots of quenching of RhEITC is evident in the temperature range of 26 to 2 9 ° C (Fig. 7). This is the temperature range where tryptophan fluorescence intensity is also discontinuous [10]. Two possibilities for the discontinuous behavior of quenching were considered: (1) the reactivity of the bound EITC changed as a function of a conformational change or (2) the accessibility of the quencher to the active site changed. Several arguments can be put forward for the second reason. The quenching mechanism is likely to be due to an electron-exchange reaction [26]. In this case, the reactivity of the excited state is related to the spectra; the emission spectra did not change over this temperature range, hence the reactivity probability also did not. The quenchers used quenched at rates considerably less than that expected for a reaction in which every collision is

effective (Table I). In spite of this, an increase in viscosity by a factor of 100 by the addition of glycerol decreased the quenching rate commensurately. The energies of activation for quenching above and below the transition temperature resembled those for the quenching of free EITC with values of 3 to 5 kcal/mol. These low values are typical of diffusion-limited reactions. Finally, in no case did the presence of quencher change the spectrum of the phosphorescence, showing that no phosphorescent complex was formed between quencher and excited-state eosin. For these reasons, it seems reasonable to conclude that the accessibility of the quencher to the probe has changed as a function of temperature. Although all the quenchers of RhEITC revealed a quenching pattern with a discontinuity in the quenching rate constant at the same temperature, details of the quenching pattern are different for the different quenchers (Fig. 7). The small quenchers, iodide and $203 (diameter about 4-5 A) show very little discontinuity, whereas methylvinylketone (linear length about 7 ,~, but flexible) shows a sharper transition. For TEMPO, (diameter of about 9 A, and nonplanar), the transition is the most pronounced. The phosphorescence quenching of eosin labeled at a site away from the active site (Rh(SS)EITC) shows no discontinuity in quenching profile (Fig. 7a). This would indicate that the transition is not due to a global unfolding of the polypeptide chain and favors the hypothesis that the quenching of EITC at the active site is responsive to domain flexibility or displacement. Another point concerning the data present in Fig. 7 should be made. It was observed that small molecules quench RhEITC with a quenching constant which is reduced only by a factor of 2 to 4 from that for free EITC. This would suggest that the amplitude of the domain motion is of sufficient size to allow the small molecule nearly free access to the active pocket. In contrast, the quenching by T E M P O was more than an order of magnitude less efficient in the rhodanese complex than for free EITC. Based upon the molecular size, we made some estimates of the amplitude of the fluctuations which would produce such a discrimination in quenching rate. The rhodanese molecule is shaped like a clam. The distance from the hinge to the opening on the other side is about

244

60 ~, [8]. Rhodanese is labeled with eosin at the active site which lies at the bottom of the crevice. Eosin is about 15 A in length. Assuming that the two halves of rhodanese open like a clam to allow penetration, we can estimate the anogle for opening required to allow a molecule of 9 A to be accessible to eosin. This angle is about 20 °. Since the large molecule, TEMPO, is considerably less efficient in quenching the protein complex than free EITC, it would indicate that the mean time leading to the transient exposure of EITC is small. The significance of a temperature-dependent structural change in rhodanese to the catalytic activity remains to be evaluated. Leininger and Westley [4] studied the temperature dependence of the rhodanese reaction between the small substrates thiosulfate and cyanide. No temperaturedependent break in the energies of activation was observed in the temperature range where we and others [10,11] see evidence for a structural change. We note, however, that the quenching constants of the small molecules to eosin in the active site did not show marked changes in temperature. Rhodanese is also able to catalyze reactions involving larger substrates [17,27]. It remains to be seen whether the binding affinities and turnover times for these substrates will reveal discontinuities as a function of temperature. In summary, a phosphorescent probe was covalently bound at the active site of rhodanese. The penetration of small molecules into the active site, as indicated by quenching of phosphorescence, is consistent with structural flexibility on the /~s time-scale. A discontinuity in the temperature dependence of quenching is interpreted as due to a structural change resulting in different domain flexibility or displacement of the domains.

Acknowledgements This work was supported by the USA National Institute of Health, grant G M 36393, and the Polish Ministry of Science, Higher Education and Technology, grant R.III.13. The authors wish to thank Dr. Gary Holtom, at the Regional Laser and Technology Laboratory, University of Pennsylvania, for his assistance in fluorescence lifetime measurements.

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