Phorochemisfrv and Photobiology Vol. 36, pp. 689 to697,1982 Printed In Great Britain. All rights reserved 003 1-8hjj/X2/12068~09$03 OO/o

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SENSITIZATION BY LUMAZINE PROTEINS OF THE BIOLUMINESCENCE EMISSION FROM

THE REACTION OF BACTERIAL LUCIFERASES

JOHN LEE Bioluminescence Laboratory, Department of Biochemistry, University of Georgia.

Athens, G A 30602. USA

(Received 3 May 1982; accepted27 July 1982)

Abstract-Lumazine protein from Photobacterium phosphoreurn blue shifts the in v i m bioluminescence spectra in the reactions using each of the I main types of bacterial luciferases: P. phosphoreum, P. leiognathi, Vibrio harveyi and V. fischeri. For the reaction initiated with FMNH, and tetradecanal at 2”C, this “sensitizing” property of lumazine protein differs quantitatively between the luciferases. An interaction constant characterizing each type of luciferase may be derived from a reciprocal plot of the spectral shift against the lurnazine protein concentration. The weakest interaction constant is in the V. fischeri reaction, 180 pM. For the V. harveyi reaction the interaction is in the range 6-9 pM, and for both Photobacterium reactions it is 2-3 p M . A concentration of only 0.6 p M of lumazine protein is sufficient to cause an observable change in the Photobacterium bioluminescence spectra. For the V . harveyi case the interaction constant is near to the equilibrium Kd for the luciferase-lumazine protein complex, observed directly by Visser and Lee. Both constants are decreased markedly by increase in phosphate concentration so that it is concluded that, with V. harveyi luciferase, sensitization occurs within this protein-protein complex. For P. phosphoreum luciferase, however, the equilibrium complex is too weak to correspond to the sensitizing interaction and it is concluded that the rate-limiting process is a protein-protein bimolecular collision. As judged from their molecular weight around 20 000, spectral properties, and ability to blue shift the bioluminescence spectra, lumazine proteins are identified in a second strain of P. phosphoreum and in P. leiognathi.

INTRODUCTION

It has recently been shown that the blue biolumi- nescence from the marine bacterium, Photo- bacterium p h o s p h o r e u m , originates from the fluorescence of lumazine protein, a novel protein accumulated in large amounts by these bacteria, evidently for this bioluminescence function (Gast and Lee, 1978; Koka and Lee, 1979; Lee and Koka, 1978; Lee et al., 1981). Lumazine protein (M, - 20000) c o n t a i n s 1 mol of 6,7-dimethyl-8- ribityllumazine, non-covalently but reversibly bound (Kd < lo-’ M, 2“, 50 mM Pi) (Small et a/., 1980; Visser and Lee, 1980). This lumazine derivative occurs naturally in procaryotes being the precursor to riboflavin in its biosynthetic pathway (Plaut, 1963). What appears to be a very similar fluorescent protein has also been found in extracts of Photobacterium leiognathi (Lee and Elrod, 1981).

These observations have led to the proposal that bacterial bioluminescence is a “sensitized” or “in- direct” type of chemiluminescence reaction (Gast and Lee, 1978; Matheson et a / . , 1981; Lee et al . , 1981). By this it is meant that the reaction can be divided into two parts, written schematically

E + S - EX

E X + A - P + A ”

where S is substrates, P products, E bacterial luciferase, and A the acceptor or “sensitizer” as it is called in chemiluminescence studies (Lee, 1977).

The first part is a sequence that forms a species of high chemical energy content designated “EX” by Matheson et al. (1981). By some mode of interaction as yet to be determined, the chemical energy of EX is transformed i n t o electronic excitation of the fluorescent A molecule. The bioluminescence is emitted in the fluorescence spectral distribution of A.

Results up to this time, including those of this present work, make it safe to generalize that for those bacteria now classified into the genus Photo- bacterium (Baumann er al., 1980), the natural acceptor in the cells is lumazine protein. In this case the second reaction in the above scheme is a protein-protein reaction. Most other types of lumi- nous bacteria are classified into the genus Vibrio and for them the identity of t h e in vivo acceptor is not known.

All bacterial luciferases, regardless of the type of bacteria, can be made to generate in virro bio- luminescence with the same substrates: FMNH?. 02, and a long chain aliphatic aldehyde, optimally tetradecanal (for a review, see Ziegler and Baldwin, 1981). In a study of the in vitro reaction with Vibrio harveyi luciferase, Matheson et al. (1981) observed a transient fluorescent species with the same spectral distribution as the bioluminescence. They proposed this fluorescent transient to be the acceptor in that reaction.

In this present work it is shown that P. phosphoreum lumazine protein can sensitize the in

689

690 JOHN LEE

vi t ro bioluminescence using any of t h e 4 well- s tud ied types of luciferase. However , t h e quant i ta - tive effect of lumazine pro te in is f o u n d t o d e p e n d o n t h e type of luciferase. All t h e results a r e explained on t h e basis of t h e above sensitization scheme. modif ied t o inc lude t h e possibility of protein- protein complex format ion between luciferase a n d lumazine protein.

MATERIALS AND METHODS

Materiuls. Lumazine protein was purified from extracts of Photobacterium by the method of Small ef al. (1980). Two different strains of P. phosphoreurn were used. strain A13 (Fitzgerald, 1978) and one from the National Collection of Marine Bacteria (Scotland) number 844. The P. Ieiognnthi was strain 477 of Reichelt and Baumann (1973) and was provided by J . Fitzgerald. For A13 growth conditions were used as described by Lee and Koka (1978) and for 844 as described by Meighen (1979). The yields of lumazine protein and luciferase were about the same in each case: 1-2 g of wet cell paste were obtained per 1 of culture iit 3 light emission level of S X 10'' photons s - ' m t - ' , corresponding to 1.5 X 10' photons s - ' cell-' based on viable count. Photohncreriurn leiognathi was grown in the same medium composition as A13 (Lee and Koka, 1978) but at 27°C using a maximum aeration rate. Both light emission per mP and yield of lumazine protein were decreased more than 3 times compared with A13. Most of the lumazine protein preparations used were purified from the next-to-last Blue Sepharose step and these were nearly homogeneous. >90% (Lee and Koka. 1978). They had no detectable luciferase activity. Authentic. 6.7-dimethyl-8-ribityllumazine was the gen- erous gift of Professor H. Wood, University of Strathclyde, and FMN (Fluka, Buchs, Switzerland) was purified by the DEAE-chromatography method of van Schagen and Miiller (1981). All other chemicals used were of the best commercial grades.

For the preparation of Photobacterium luciferases the procedure of Gast et al . (1978) was modified. Standard buffer contained phosphate (50 mM. pH 7) and all buffers contained 2-mercaptoethanol (2-5 mM); all procedures were carried out at 5°C. Following ammonium sulfate fractionation (30 and 80%) of the cell extract (200 g wet cell paste) and desalting on Sephadex (3-75 (Pharmacia. Piscataway, NJ) . the luciferase fraction was loaded to a DEAE-Sephacel (Pharmacia) column ( 2 X 30 cm) pre- washed with the standard buffer except at pH 7.6. The column was then washed with this same buffer (500 me, pH 7.6). then with 100mMphosphate (500mY, pH7.6) and the luciferase "hatched" off with 150 mM phosphate (500 me, pH 7.6). Some separation of the "luciferase-associated" lumazine protein. which preceded the luciferase, some- times occurred at this point. The combined luciferase fractions were concentrated to 100 ml by ultrafiltration on a PM-30 membrane (Amicon Diallo. Lexington. MA), then loaded onto a column (2 x 20 cm) of DEAE-Sephadex-A50 (Pharmacia), prewashed with phosphate buffer (120 mM. pH 7.6). Elution was continued with the same buffer (1 Z). and subsequently with 150 mM phosphate (pH 7.6, 1-2 1 ) . The luciferase fraction, practically free of lumazine protein by this stage, was concentrated to 15 ml by ultrafiltration. then subjected to high resolution gel chromatography on either Ultrogel AcA 44 (LKB, Rockville, MD) (3.7 X 90 cm. 40 mPih) or Sephacryl S-200 (Pharmacia) (3 X 80 cm. 40 mtih), eluting with standard buffer. pH 7.0.

The luciferase at this stage is homogeneous as judged by SDS-acrylamide gel electrophoresis and it has high specific activity. For P. phosphoreum luciferase this is typically 1.5 X lo'-' photons s ' ~' with dodecanal at room temperature: P. leiognathi has more than twice this specific

activity. Tetradecanal is the aldehyde giving the highest specific activity: e.g. for A13. 1.4 X 10" photons s ' A2& ' . The activity is stable for months when stored at -10°C in buffer containing 20% glycerol. The above procedure produces about 0.5 g of purified luciferase in about 30% ocerall yield. A flavoprotein-like impurity occurs in these luciferase preparations at a level of 5-10"/0 on a molar basis (Gast et al.. 1978). This impurity is very difficult t o remove and may only be reduced to 5'1; (A2xlJ/A4511 - 150) by chromatography on D E A E - Sepharose (20 x 2 cm), washing with 100 mM P,. pH 7.6. SO0 mP, then eluting with 130 mM P,. pH 7.6. Most of the preparations used had A18,1/A45,1 ratios in the range 50--80.

Luciferases from Vibrio (formerly Phorohucreriitrn) fifischeri. strain 399 and V . (formerly Beneckeu) harveyi. strain 392 (Reichelt and Baumann. 1973: Baumann er 01.. 1980) were prepared by similarly modified procedures. as described above (Gast et (11.. 1978). except the second ion exchange step utilized DEAE-Sepharose. pH 7. The preparations were homogeneous and had much lower (- 1 "%) flavoprotein-like impurities. Further reduction of this contaminant was achieved by concentrating the DEAE- Sepharose fractions to 15 mC. loading onto a 3.5 X 70 cm column of Blue Sepharose (Lee and Koka, 1978) and eluting isocratically with standard buffer.

The NADH:FMN oxidoreductase used was prepared from V . fischeri 399 by a method modified from Puget and Michelson (1972: White, 1980). N o attempt was made to purify it to homogeneity but only to obtain enough activity (1-2 +mol N A D H min- ' AZXI;') to provide the coupled bioluminescence described below. It was rela- tively free of the flavin-like contamination (A2KI)IA45,, = 15(&200) and had no luciferase activity.

Methods. Thc luciferases were assayed at room tem- perature by rapid addition of FMNH? (0.5 mC. photo- reduced from FMW, 80 p,M in 50 mM P,, pH 7 buffer containing EDTA. 20 mM) into the luciferase sampk ( 1 m t ) in standard buffer containing BSA ( I mgimt) and dodecanal (10 K P . methanol-saturated solution). For V . harveyi luciferase, decanal was used (10 p,e, 5% saturated solution in methacol). The bioluminescence spectra were measured in a similar FMNH, reaction. except at 2°C usually in a total volume of 0.35 ml with the added reagents as described. Alternatively. an NADH-FMN coupled system was employed where NADH was added to the reaction mixture (0.35 mC final volume) containing FMN, aldehyde and an amount of purified oxidoreductase from V . fischrri to generate the desired light level.

Absolute photometric calibration was obtained by reference to the NBS Standard Lamp via the luminol chemiluminescence reactions (Lee et al.. 1966). Emission spectra were measured on an instrument previously des- cribed (Wampler. 1978). The bioluminescence intensity changes rapidly during the time of the spectral scan. more so in the FMNH? rcaction than in the oxidoreductase coupled reaction. The signal at each wavelength. there- fore. is presented as a ratio to the total intensity obtained from a rcference phototube viewing the undispersed total emission from the sample. This is the "charge-normalira- tion" technique of Seliger (15'60). The spectra obtained are. therefore. normalized to a constant area. Data collection was made on-line with a Nova computer and the SPECOS software generously provided by Dr. J . E. Wampler (University of Georgia). Its operation is briefly described elsewhere (Wampler el a / . . 1979; Wampler. 1981). All spectra represented have been corrected for instrumental distortion by reference to an NBS Standard Lamp, and for self-absorption (Wampler. 1978). Reactions were carried out in special cells of short path-length. 2 or 1 mm, to minimize the self-absorption correction factor. The optical density was measured directly on the reaction product or was calculated as the sum of contributions from added protein and FMN, with the assumption that they

Lumazine protein bioluminescence 69 1

i J 2 - 0 m I .~

t

+

4c0 5 x 530

WAVELENGTH (nm)

Figure 1. The addition of lumazine protein from P. phosphoreum shifts the bioluminescence spectrum for the in virro reaction using 4 different types o f luciferases: (a) P . phosphoreum. (b) P. leiogrzarhi. (c) V . harveyi and (d) V . fischeri. The numbers on each spectrum are the micromolar concentrations of lumazine protein: the unlabelled one has none added and the dashed spectrum is the one with the highcst concentration. All reaction conditions are the same: FMNH, 10 F M , tetradecanal SO p.M. luciferase

protein concentration 1&1S p M . in 50 mM P,, pH 7 and 2°C.

were both in the fully oxidized states during t h e coursc o f the bioluminescence.

To determine the concentration of lumazine protein. it was assumed that the lumazine group was bound in a 1 : 1 stoichiometric ratio to the protein and that the molar decadic extinction coefficient for the bound lumazine group at its absorption maximum. 414 nm (Small er 01.. 19x0: Visser and Lee. 1980: Koka and Lee. 1981). was the same as that of the free molecule in aqueous solution. ~ ( 4 0 7 ) = 10300 M - ' c m - I (Malcy and Plaut. 1959). For the luciferases. protein concentrations were calculated o n a molar basis usins the literature values of qxcific cxtinction coefficients and mol wt: P. phosphoreum luci- ferase. 1 .1 m t mg cm I. M , 82000 (Yoshida and Nakamura. 1973); V. fischeri and V . hurveyi, 0.94 ml mg I cm I, M , 79000 (Gunsalus-Migucl et a/.. 1972); P. leiognarhi luciferase was assumed to be 1.0 mt mg I

cm- I. M , 80000. Absorption spectra were measured with a Cary 14 spectrophotometer.

RESULTS

Figure 1 shows that the addition of P. phos- phoreum lumazine protein to the in vitro biolumin- escence reactions, using 4 different types of bacterial luciferase, shifts the spectral maxima to shorter wavelengths. Except for the V. fischeri reaction (Fig. Id) the spectra are observed to change in an isoemissive manner. All reactions are carried out under identical conditions as described in the legend of Fig. 1.

Figures 1 a and 1 b are for the reactions using each of the two Photobacterium luciferases. Similar results to Fig. l a have been reported previously but under different reaction conditions, using the oxidoreduc- tase system to generate the FMNHz and at 12°C (Gast and Lee, 1978: Lee et al . , 1981). It is the lower temperature of 2°C that enhances the effect of lumazine protein in the present case. For both luciferases (Fig. l a and b), the effectiveness of

lumazine protein for blue shifting the spectral maximum is very similar. With 21 IJ-M lumazine protein (not shown; cf. Lee et al. , 1981) the bioluminescence spectrum with P. phosphoreum luciferase has an identical spectral distribution to the fluorescence of lumazine protein (max 472 nm). For P. leiognathi, however, the maximum remains at 474 nm as in Fig. l b and is broader than the fluorescence of lumazine protein.

Figure l c shows that for V . harveyi luciferase under the same reaction conditions. a several times higher concentration of lumazine protein is required to produce the same spectral shift as for Photo- bacterium bioluminescence. However. the spectral maximum for V. harveyi bioluminescence at 487 nm is 8 nm to the blue of the Photobacterium reactions (495 nm). It is also observed in Fig. Ic that the maximum shift of the V. harveyi bioluminescence brings it only to a 477 nm maximum. and this is distinctly ( 5 nm) to the red of lumazine protein's fluorescence. Higher concentrations of lumazine protein (not shown) produced no further blue shift.

Figure Id is the bioluminescence with V. fischeri luciferase. The concentration of lumazine protein required to produce an observable change in this bioluminescence spectrum is more than an order of magnitude greater than for the Photobacterium reactions. Because it is technically difficult to obtain precisely corrected spectra with lumazine protein at very high concentrations, no attempt was made to determine the maximally shifted spectrum by work- ing above the 80 k*.M shown here.

Putative lumazine proteins were isolated from two other types of Photobacterium. The first type was a different strain of P. phosphoreurn, NCMB 844. The fluorescent protein purified from these extracts had a mol wt around 20000 by gel filtration, and identical fluorescence and absorption spectra to

692 JOHN LEE

w 0 P le,ognathi I

433 500

WAVELENGTH (nm)

Figure 2. The addition of a putative lumazine protein from two other types of Photobacterium shifts their in uirro bioluminescence spectra. Upper panel: a different strain of P. phosphoreum. NCMB844. is used, with its own luciferase and lurnazine protein. except for the dash spectrum to demonstrate the cross reaction with 34 p M of lumazine protein from strain A13. Also the reactions are made using t h e o x i d o r e d u c t a s e e n z y m e t o g e n e r a t e t h e FMNH2:luciferase 15 p M , dodecanal 50 p M . FMN 3 p M . NADH 250 p M . 50 m M P, buffer. pH 7 and 12°C. Lower panel: a different species, P. kiognarhi, is used with and without its own lumazine protein. under the same reaction

conditions as in the Fig. 1 legend.

the lumazine protein from strain A13 (results not shown). Figure 2, top panel, shows that the addition of this NCMB 844 fluorescent protein to the bio- luminescence reaction using NCMB 844 luciferase, blue shifts the spectral maximum. The dashed spectrum was with 34 p M of the lumazine protein from strain A13 to demonstrate the cross-reaction. The overall result is exactly the same as for Fig. la . Also in this reaction the FMNHz was generated from N A D H via the NADH:FMN oxidoreductase. so this experiment also demonstrates that the method of introducing the FMNH2 makes no difference.

A lumazine-like protein has also been partially purified from P. leiognathi (Lee and Elrod. 1981). In Fig. 2. lower panel, it is seen that addition of this protein to the bioluminescence reaction using P. leiognathi luciferase shifts the spectral emission maximum to the blue. This protein is equally effective for the reaction using P. phosphoreunz luciferase (results not shown) corresponding to the reverse cross-reaction of Fig. l b .

The spectral shift of the bioluminescence maxi- mum induced by concentrations of lumazine protein can be described in quantitative terms by making use of a double reciprocal relation

A h R - ’ = AhM-’ (1 + K.LumP-’)

where Lump is the micromolar concentration of

lumazine protein and K is an experimental inter- action constant: the spectral shift is A X B = h , , - h ~ . where A,, is the bioluminescence spectral maximum in the absence of lumazine protein and hB that with the lumazine protein; A X M is the maximum spectral shift. The constant K is just that concentration of lumazine protein required to produce one half the maximum spectral shift and is a measure of the “effectiveness” of the lumazine protein.

In Fig. 3 the reciprocal relation is plotted for all the data obtained from the bioluminescence reac- tions using Photobacterium and V. harveyi luci- ferases. The results for the two species of Photo- bacterium luciferases fall on the same line and the value of the interaction constant, K = 2.5 k 0.3 p.M. Two preparations of V . harveyi luciferase were studied which had different flourescent spectral impurities as noted by Matheson and Lee (1981). For 350 nm excitation, one preparation (A, Fig. 3) had a fluorescence maximum at 430 nm. and the other (0) at 490 nm. The shifts for V. harvevi bioluminescence a r e smaller than for Photo- bacterium and so the points are more scattered. No difference can be distinguished between these two preparations of V. harveyi, but it is clear that the line fitted to the combined data yields an interaction constant, K = 8 k 2 K M , significantly greater than the one for Photobacterium.

[LurnPl-’ (rnM-”i

Figure 3. Reciprocal plot of the shift (AX,) of the in tmirro bioluminescence spectral maximum on addition o f concentrations of lumazine protein (Lump). Reaction conditions are as in the legend of Flg. 1 with the luciferases from P. phosphoreum ( X ) . P. leiognarhi (0). and two different batches of V. harveyi ( 0 . A ) . Thc two lines arc obtained by a linear least squares fit to the data from both Phorobacferium luciferases in one case. and to the two V . harueyi luciferases in the other. The interaction constant, K , is evaluated from the intersection of the line on the

abscissa.

Since only two points are available for V. fischeri (Fig. Id). the reciprocal plot is not made. Assuming the same A A M as for V. harveyi, the interaction constant is estimated as approximately 180 p M .

Figure 4 demonstrates that phosphate concen- tration has a strong influence on the interaction constant of lumazine protein in the V. hanseyi bioluminescence. The reactions were run at room

693 Lumazine protein bioluminescence

50mM P,

0 5 r 1

m M P 8

1. ~~ L - L - L . L - L U 100 300 500

[LurnPl-’ ( mM”)

Figure 4. Phosphate concentration has a strong influence on the bioluminescencz spectral shift (Ahs) induced by P. phosphoreum lumazine protein (Lump) in the in v i m reaction using V. harveyi luciferase. Reaction conditions are as in the legend of Fig. 1 except with dodecanal and at

room temperature.

temperature to compare the interaction constants with the protein-protein dissociation constants at room temperature, measured by Visser and Lee (1982). Dodecanal was used instead of tetradecanal to slow the decay of the bioluminescence intensity and provide bet ter precision of the spectral measurement. It was found that for the reaction at 2°C using dodecanal and V. harveyi luciferase, the spectral shift induced by lumazine protein was identical to that for the reaction with tetradecanal.

In a solution containing 200 mM PI (Fig. 4 , O ) the interaction constant of lumazine protein in the bioluminescence using V. harveyi luciferase is K = 4 pM at room temperature. This result is comparable with the interaction constant with P. phosphoreum luciferase at 2°C. However, in this latter case, phosphate concentration has no effect on the spec- tral shift, nor has phosphate concentration any effect on the ho obtained with any of the 4 luciferases.

In 50 mM PI buffer the interaction constant measured at room temperature for V. harveyi is K = 12 pM, a factor of 1.5 times greater than the interaction constant at 2°C. For the Photobacterium luciferases the effect of temperature on the inter- action constant is somewhat greater; more than a 3-fold increase to K = 10 pM is found at room temperature.

Figure 4 shows that in 200 mM PI, the highest concentration of lumazine protein used shifts the spectral maximum again to only 477 nm (cf. Fig. 1 c). This spectral distribution can be duplicated by a mixture of 60% contribution from lumazine pro- tein’s fluorescence and 40% from the biolumin- escence spectrum without addition of lumazine protein (Ao = 487 nm). If the fluorescence of lumazine protein in the protein-protein complex was red-shifted over free lumazine protein, then this

400 500 600

WAVELENGTH (nm)

Figure 5. The fluorescence spectrum of P. phosphoreum lumazine protein is not changed in its complex with V . harveyi luciferase. The upper spectrum is lumazine protein (15 F M ) slightly displaced from the same in the presence of Iuciferase (240 FM protein), lower spectrum (luciferase contribution subtracted). Solution is 50 mM Pi, pH 7. 2°C;

excitation 418 nm.

would account for this result. Figure 5 compares the fluorescence of lumazine protein alone and in the complex with luciferase (Visser and Lee, 1982). The two fluorescence distributions are identical.

Figure 6, lower panel, plots the position of the bioluminescence spectral maximum in the presence of lumazine protein against the luciferase concentra- tion. Two cases are given; for P. phosphoreum luciferase ( x ) and 1.8 p M lumazine protein, the spectral maximum remains constant at 483 nm. The same constancy is foun: with other lumazine protein concentrations, with dodecanal or in the reaction using the oxidoreductase coupled system t o generate FMNHz. The open circles are for the V . harveyi bioluminescence but with 3.5 p M lumazine protein and in 200 mM Pi. Again there is no change in spectral maximum with increase of luciferase concentration.

The concentration of added FMNHz was found to be a factor which does influence the spectral maxi- mum. Under the conditions of Fig. 6, lower panel, using 15 pM luciferase, the spectral maximum is at 488 nm if 18 pM FMNH2 is used in the V. harveyi reaction. The same shift requires 100 pM FMNHz for P . phosphoreum.

Since it is known that under similar reaction conditions a major fraction of the FMNH2 is rapidly oxidized to FMN (Lee and Murphy, 1973), it was presumed that this FMNH2-shift might be due to FMN, and the upper panel of Fig. 6 confirms this supposition. For the V. harveyi reaction in the presence of 3.5 p.M lumazine protein (0), the spectral maximum shifts to longer wavelengths proportional to the FMN concentration. The lines are least-squares fits. The open square is for a 88 p M concentration of FMNH2 instead of the 10 p M used for all the other reactions. FMN concentration also affects the hoof this reaction in a like manner (0; Fig.

JOHN LEE

LUCIFERASE CONCENTRATION ( p M )

Figure 6. Uppcr panel: the presence of FMN in the reaction shifts the bioluminescence spectral maximum to longer wavelengths. (All spectra have been corrected for self-absorption. sec Methods.) The reaction conditions are as in thc legend of Fig. 1 using V. harveyi luciferase (0). In two reactions riboflavin was used instead of FMN (A; ., 1 mm path). The (x) are with FAD instead of FMN. The point5 (0) are with lumazine protein (3.5 p M ; 200 mM P,) and (0) is using no FMN but 88 pM of added FMNH, instead of the 10 pM used in all the other reactions. Lower panel: increasing the concentration of luciferase has no effect on the spectrum in the presence of lumazine protein: (0) V . hurwyi luciferase with lumazine protein 3.5 p M . 200 mM P,: ( X ) PI phosphorrum with lumazine protein 1.8 p M , SO mM P,; other reaction conditions as in the legend

of Fig. 1.

6. upper panel); the two least squares fitted lines are parallel. The shift is not specific for FMN as riboflavin and F A D display the same behavior (A and x, respectively). The square point is for riboflavin in a 1 mm pathlength as a check on the corrections for self-absorption; this small discrep- ancy between the two estimates of A B would be expected to be much less at lower concentrations, as the correction factors become much smaller.

The spectral shift induced by all the flavins used is accompanied by a strong reduction in the bio- luminescence light yield. At the highest concentra- tions used in Fig. 6 (upper panel), the light is reduced about 20 times. As well the decay rate of bioluminescence intensity is increased about 3-fold.

The concentration of FMN also red shifts the An and A n for the P. phosphoreum bioluminescence, but, consistent with the relative effect of FMNH2 noted above, the FMN is about 5 times less effective than in the V. harveyi reaction. No attempt is made here to obtain more than this semi-quantitative result. The concentrations of FMN that would be

Figure 7. Lumazine protein shifts the spectral maximum o f the in vifro bioluminescence using V. htrrrmeyi luciferase and reduced lumichrome (15 p M ) : other reaction conditions are as in the legend of Fig. 1. (0) shows the presence of

200 mM P,.

required are too high for corrections for self- absorption and other artifacts to be reliable.

Lumazine protein also blue shifts the biolumin- escence using reduced lumichrome in place of FMNH2. Figure 7 is the reciprocal plot for the reaction with V. harveyi luciferase. The batch of luciferase used for this experiment is one that exhibited the same A,, with reduced lumichrome as FMNHz (Matheson and Lee, 1981). The interaction constant, K - 8 pA4, in Fig. 7 is the same as for the FMNH2-bioluminescence under the same experi- mental conditions. The filled point in Fig. 7 is for a higher phosphate concentration. No significant change in AXB is evident

DISCUSSION

When Gast and Lee (1978) first reported that lumazine protein added to the P. phosphoreum in vitro reaction shifted the bioluminescence spectrum to the blue so that it became identical to the in vivo bioluminescence distribution, accompanied by a change in the bioluminescence kinetics and an increase in the light yield, there were two clear implications. The first was that the emitting mole- cule in the cell was this protein-bound lumazine derivative. This proposal is now reinforced by the findings of this present work that, based on spectral and bioluminescence properties, lumazine protein is f o u n d i n o t h e r b a c t e r i a within t h e genus Photo bacterium.

Lumazine protein bioluminescence 695

The second implication was that the energy could not be transferred from whatever the emitting molecule is in the in vitro reaction to lumazine protein. This would be an energetically uphill process and unlikely to occur efficiently (Muller. 1981). Lumazine protein has its visible absorption maximum at 416 nm and the in vitro bioluminescence emitter has a spectral maximum at 495 nm, and its distribution has little spectral overlap with the absorption of lumazine protein. As a consequence the reaction of the substrates with luciferase has to generate a primary energy source of sufficient energy, around 300 kJimol, equivalent to a wave- length of 400 nm, to populate the first singlet excited state of the lumazine or, in its absence in the in vitro reaction, the fluorescent state of the unidentified acceptor emitting at 495 nm (Lee et al., 1981; Ziegler and Baldwin. 1981). The presence of some emitting species of this high energy has recently been substantiated by Matheson et a[. (1981).

The main findings of this present work are that lumazine protein is capable of accepting the excita- tion energy in the bioluminescence reactions using any of the 4 main types of bacterial luciferases, but that its property in this respect differs quantitatively among the luciferases. These are the 4 most studied of the bacterial luciferases and they are known to also differ significantly in some of their other properties (Ziegler and Baldwin, 1981). Another result is that the interaction constant of lumazine protein does not differ quantitatively, whether FMNHz or reduced lumichrome is used as the reduced substrate. This implies that the rate limiting step in the transfer of energy into the lumazine is independent of the chemical nature of the reduced substrate.

All these results are accounted for by introducing a protein-protein complex into the scheme des- cribed in the Introduction (Scheme I) . Two modes of transformation of the chemical energy of E X

E E X L P + A *

\ ELumP EXLumP- P + Lump*

occur, either a bimolecular process in which lumazine protein and A are competitive, or an intramolecular process (lower) in which only the complexed lumazine protein is excited. The possi- bility of an equilibrium complex formed by reaction of EX and Lump is also consistent with the present data.

Visser and Lee (1982) have provided direct evidence for the formation of a complex between native luciferases and lumazine protein, by measur-

PAP 36.6 - F

ing the relaxation rate of the emission anisotropy of the bound lumazine. For V. harveyi luciferase. 3°C. 50 mM Pi. pH 7, the complex has an equilibrium K d = 2-3 p M . They additionally observed that this luciferase was not homogeneous in its interaction. typically 50% of the luciferase protein was “incompetent” for the complex formation.

The interaction constant K in the present set of experiments is a kinetic parameter, not an equi- librium one. It is a measure of the relative rate- limiting steps of reaction of EX via the “A” pathway vs the “LumP” pathway(s). The fact that for V. harveyi luciferase (Fig. 3) K is only a few times higher than K d is suggestive that they are related. In fact by allowing for an amount of incompetent luciferase they become nearly the same. Therefore. we conclude that the complex is functional and the energy of EXLumP is transferred directly to the complexed lumazine. Further support for this idea is that increase of phosphate concentration decreases K (Fig. 4) paralleling the effect of phosphate concentration on Kd (Visser and Lee, 1982).

Uncomplexed luciferase, both competent and incompetent, produces EX, and lumazine protein competes with A for this energy in an inter- molecular process. This explains why at high luma- zine protein concentrations the bioluminescence is only shifted to 476 nm (A&’ - 0.1 nm-I; Figs. 3 and 4) where the spectral distribution can be constructed from about equal contributions from lumazine protein fluorescence (via EXLumP) and the 487-nm species (EX -t A).

The supposition that a rapid equilibrium also exists between E X and EXLumP explains why the spectral shift does not depend on the concentration of E . The added FMNHz is constant and limiting so the amount of E X formed does not depend on E in the concentration range E > FMNH? (Fig. 6). The ratio EX/EXLumP depends only on the presence of Lump, to a rough approximation. If this second equilibrium did not exist then as E was increased, a greater proportion of the FMNHz would be con- verted to E X and the spectrum woufd shift to longer wavelength, via E X + A .

Only a weak complex was observed to be formed between lumazine protein and P. phosphoreum luciferase (Kd < 900 p M , 3”C, 50 m M Pi, pH 7 ; Visser and Lee, 1982). Yet under the same reaction conditions lumazine protein is more effective at sensitizing the bioluminescence with P. phospho- reurn than with V. harveyi luciferase. Even a 0.6 FM concentration of lumazine protein has a noticeable effect on the P. phosphoreum bioluminescence spectrum (Fig. la) . If no complex is present, then the sensitization by lumazine protein must be by the bimolecular process in the scheme, i.e. E X + Lump. This means that while lumazine protein may efficiently sensitize the bioluminescence within a protein-protein complex, such a complex is not necessary for efficient sensitization.

L t f

Visser and Lee (1982) were unable to observe any complex between V . fischeri luciferase and lumazine protein, The spectral shift is real (in Fig. Id) and cannot be attributed to trivial absorption and re- emission o f the fluorescence of lumazine protein. The interaction in the V . Jischeri reaction is. there- fore. proposed to be similar to that in Photo- bacteritrrn. where the lumazine protein competes with A in a bimolecular reaction. In such a process, the different effectiveness of lumazine protein with each of the luciferases is a reflection of the competi- tiveness of A. involving among other things. the steady state concentration of A and its fluorescence quantum yield. Therefore. a brief discussion of the nature o f A is appropriate, leaving a more detailed account for ekewhere.

Matheson e t a / . (1981) have suggested that, in the V. hrmyyi reaction, A is the fluorescent transient species formed in the course of the bioluminescence process. Kinetic evidence suggests that this fluores- cent transient intercepts the energized species E X in a protein-protein reaction analogous to the E X + Lump reaction in the above scheme (Matheson and Lee, 1982). In fact, one might generalize A to include lumazine protein or any orher fluorescent species present in the system, provided it qualifies energet ical ly . Consequent ly , in the reduced lumichrome bioluminescence reaction, the fluoro- phores that accompany the luciferase preparation at low level as well as under certain conditions. the oxidized product, lumichrome. all can be excited (Matheson and Lee, 1981).

The addition of another fluorophore, FMN, shifts the bioluminescence to the red in Fig. 6. since it competes with the other acceptors and adds its fluorescence, 535 nm maximum (Eley et af., 1970) to the total. This shift is accompanied by a strong reduction of the light yield since the luciferase quenches the fluorescence of FMN (Ziegler and Baldwin. 1981) and probably riboflavin as well. The FAD reduction accompanying the shift is more likely due to the fact that its fluorescence yield is low.

Thc luciferases other than V. havveyi have not been studied with respect to a similar fluorescent transient. but one is expected to occur. Then the high effectiveness of lumazine protein with P. phosphoreuni must be explained as due to a low steady state level of the transient or by a specific interaction between EX and lumazine protein. For the competent for complex formation V . hurveyi luciferase. lumazine protein out competes any other acceptor by virtue of its propinquity. For the other Vibrio luciferase, V . Jischeri, lack of any compe- tence for complexation requires the lumazine pro- tein to compete on an equal footing with the putative fluorescent tranient formed with that luciferase.

Finally. Fig. 7 shows that the use of reduced lumichrome in place of FMNHz in the reaction with

V . hurvryi luciferase makes little difference to the lumazine protein interaction constant. This is con- sistent with the major part of the bioluminescence going via the complex EXLumP. the same complex as formed starting from FMNH:. and having the same distribution between free and bound EX.

It is safe to generalize that lumazine protein is the common emitter in all Photobacterium since it is found effectively cross reacting between 3 different types. The question arises why lumazine protein does not form a strong complex with its comple- mentary luciferase when it forms a tight. specific and functional complex with V . /iari,eyi luciferase? The high yields of Iumazine protein which can be extracted from P. phosphorrum (Lee and Koka, 1978) suggest that the intracellular concentration is very high and perhaps other acceptors are out- competed on this basis alone. Alternatively. the observation that V . hurveyi luciferase is hetero- geneous in its ability to form complex possibly could be the result of some preparation artifact. e .g . deamidation. The other luciferases may be more prone to this problem losing during purification practically all ability to form the complex but retaining the bioluminescence potential. Isolation of nascent complexes from all these cells should be feasible and in fact, the existence of a multi-protein complex of luciferase in extracts of V . ,fischeri ha5 recently been claimed (Danilov. 1979; Danilov and Egorov. 1981).

Based on the identity of their emission spectra the acceptor in vivo in V . hnrveyi, strain MAV. is probably the fluorescent transient. However. the tightness. specificity and functionality of the com- plex formed between this luciferase and lumazine protein suggests that i f the organism were to need an emission further to the blue, accumulating lumazine protein would achieve this. Strains of V . harvevi emitting in vivo near to a 480 nm maximum are available (Fitzgerald, 1978) and attempts to identify a lumazine protein in these are in progress. All these results point again to a certain versatility of the luciferase: it will use any qualifying emitter i t can find (Matheson et a/.. 1981).

Acknowledgements-I thank I-Wen Hsu. Martha G. Elrod, Bruce Gibson and Harold Benton for their technical assistance at various stages of this project: James Linn and Dr. Ron Makula for operating the fermentation facility; Dr. E. A. Meighen, McGill University for supplying the strain NCMB 844; Dr. J . M. FitzGerald. Swinburne Institute of Technology, Australia, for the strain 477; and Dr. J . E. Wampler, University of Georgia for the software used for much of the data analysis. This work was supported by NSF PCM 79-11064 and NIH GM 28139.

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