Bacterial bioluminescence: Absorption and fluorescence characteristics and composition of reaction products of reduced flavin mononucleotide with luciferase and oxygen

Vol. 53, No. 1,1973

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BACTERIAL BIOLUMINESCENCE: ABSORPTION AND FLUORESCENCE CHARACTERISTICS AND COMPOSITION OF REACTION PRODUCTSOF REDUCEDFLAVIN MONONUCLEOTIDE WITH LUCIFERASE ANU OXYGEN John Lee and Charles

L. Murphy

Department of Biochemistry University of Georgia Athens, Georgia 30602 Received

May 15,

1973

Summary: The initial reaction products of FMNH2,oxygen and bacterial luciferase are a flavoprotein, free FMNand hydrogen peroxide. This flavoprotein then adds a mole of oxygen to give a product which either reacts with a longchain aldehyde to give bioluminescence, or in the absence of aldehyde decays to free enzyme, free FMNand hydrogen peroxide. Quantumyield measurementsand chemical identification established the in vitro

reaction of bacterial

2FMNH2+ 202 + RCHO % This is a luciferase aliphatic

(E) catalyzed

intermediate

of the light

which either

with the MAV luciferase.

order manner (4).

the half-life

or if

aldehyde

This intermediate

is sta-

being about seven minutes at 5'C

Wehave taken advantage of this fact to characterize

products of the reaction

Two steps in the reaction in the formation of the initial

of oxygen with FMNH2on the enzyme by of oxygen and appearance of FMNand Hz02.

of oxygen with FMNH2and enzyme may be discerned products, the first

The FMNHzis completely oxidized

equivalent

the other

as the formation of a long-lived

reacts with aldehyde to give light

determining the rates of utilization

ond.

of FMNH2and a long-chain (N>8)

emission obtained diminishes in propor-

This has been interpreted

at low temperature,

the initial

oxidation

in the emission of light.

is absent decays away in a first bilized

U-3)

of aldehyde to the reaction is delayed after

components, the intensity tion to the delay.

bioluminescence:

2FMN+ Hz02 + Hz0 + RCOOH

aldehyde resulting

If the addition

have recently

in the first

much faster

than the sec-

step by only one-half

of oxygen to form one-half equivalent HzOz. The enzyme is

Copyright 0 I973 by Academic Press. Inc. All rights of reproduction in my jixm reserved.

157

Vol. 53, No. 1,1973

apparently

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reduced in this step since one-half of the FMNHzinjected

as free FMN, and the other half bound to form a flavoprotein.

is found

This flavopro-

tein adds oxygen in the second slower step to form an oxygenated intermediate which can break down (t+ 7 min) to form free enzyme, FMNand HzOz. EXPERIMENTAL Luciferase from the bacterium type "MAV" (5) was prepared by the method previously

reported for the purification

(1) with the additional The luciferase

of Photobacteriwn

step of elution

fischeri

from DEAE-Sephadexby a linear

appeared homogenousby the normal criteria

resis and velocity

sedimentation.

Fluka AG, and further

purified

All spectra and reaction

gradient.

of disc electropho-

FMN (86% nominal purity)

was purchased from

by DEAE-cellulose chromatography.

chemicals were of the best available

luciferase

All other

reagent grade.

rates were measured at 5'C.

Absorption spectra

were determined on a Cary 14 spectrophotometer and fluorescence on an instrument previously

described (6).

the use of Polaroid filters

Fluorescence polarization

with this instrument.

determined with a TRWnanosecond lifetime

was determined by

Fluorescence lifetimes

instrument.

Oxygen consumption was followed by a Clark oxygen electrode Cambridge, England) calibrated

were

as previously

was determined by measuring the difference

described (1).

(Rank Brothers,

Hydrogen peroxide

in oxygen consumption in the pres-

ence of catalase (1). RESULTS WhenFMNHzis added to luciferase

in air-saturated

solution the autooxi-

dation competes with the reaction with enzyme: E-FMNHz +

light

FMN+ H202 Since the autooxidation

is fast

(7) and the overall 158

rate of light

emission

BIOCHEMICAL

Vol. 53, No. 1, 1973

far

slower

compete

(4),

oxygen

sufficient

for

luciferase

vitro

in

QB(FMNH2)

reacts

with

the

is

observed

to reach

at this

concentration

lowed

time

from

yield

long-lived

a constant

interval

quantum

yield

and above

with

a tt

respect

This

formed

when 4.0

of luciferase of decanal

was found

of seven

found

of 3% in the

(2).

by injecting

by injection

2

has been

com-

maximum

to 0°C.

solution

QB(FMNH2)

to out-

with

value

intermediate

an air-saturated

concentration

of bioluminescence

and FMNHz was determined

time

interval

quantum

room temperature

of the

into

at a known

The relative the

rate

cc)

of 40 uM (3 mg/ml)

since

oxygen

FMNH2 (0.1

A concentration

this

is unchanged

The decay

in a high

FMNH,.

reaction

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must be present

for

to FMNH2, QB(FMNHz), plete

AND BIOPHYSICAL

(0.1

to decrease

luciferase

or 8.0 nmoles

of

(40 uM, 1 cc)

fol-

cc,

aqueous,

400 PM).

logarithmically

with

minutes.

Fluorescence On addition fluorescent

of FMNH2 to an air

product

spectral

appears

distribution

remains

constant

FMNH2 were

fully

of fluorescence

these

were

ded that

minutes

oxidized

one-half

rest

must be bound

this

luciferase

and free

added

measured from

which

[5“C),

expected

a

has a fluorescence

The fluorescence

intensity

if

all

The fluorescence

of the

lifetime

added and

to be 651 nsec and ~2% respectively; free

FMN.

FMNHz is now oxidized

to some form

show that

of FMN.

in solution.

were

of luciferase

30 seconds

at 50% of that

indistinguishable the

solution

the first to that

several

polarization values

within

identical for

saturated

of luciferase.

FMN fluorescence

From this and free

Control is

not

data

it

in solution

experiments

quenched

is

conclu-

while with

the

FMN and

in the presence

of

MAV luciferase. Absorption Several nm is

found

The total products

minutes

after

the

to be 84% of that absorption

spectra

are shown

in figure

180 seconds)

has absorption

addition expected

of the 1.

of FMNH2, the optical if

all

initial

The initial

maxima

of the reaction absorption

density

FMNHz were mixture

fully

oxidized.

and of the

spectrum

final

A (60 < t <

at 445 and 370 nm, the same as for

159

at 445

free

Vol. 53, No. 1,1973

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

400 WAVELENGTH

0

I 500

450 Cnml

Fig. 1. Absorption spectra of reaction intermediates formed on injection of FMNHg into a solution of luciferase and oxygen. Curve A is the initial absorpCurve B is the spectrum obtained on tion spectrum (60 set < t < 180 set). subtraction of an FMN spectrum corresponding to one-half the FMNH2 injected from curve A. Curve C is the final product (t >1200 sec.) and corresponds to free FMN. FMN except than Since

that the

subtraction the

amount

corresponds with

that

the

expected

370 nm absorption if

100% of the

fluorescence

results

of a free

a strongly

from

non-fluorescent

enhanced

considerably

FMNHp injected

have

FMN spectrum

of FMNH2 added to the

is

curve

shown that equal

enhanced

were half

present the

to 50% of the

A, results

in

curve

FMN and can be seen

370 nm maximum which

160

taken

and greater as free

FMN is total B.

fact

expected This

to have

together

in

FMN. free, for

spectrum

an abso-option

with

the

absence

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Vol. 53, No. 1,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

120

I

600

360 t/ set

and O.D. 445 appearance on injection of Fig. 2. Rates of oxygen utilization FMNHzinto a solution of luciferase and oxygen. The triangles (A) represent the appearance of O.D. 445 as a fraction of the final O.D. 445. The open circles represent the 02 consumedas a fraction of the final O2 consumption. The semilog plot (insert) shows the psuedo first order reaction of the intermediate with oxygen. The Y axis (1-Z) is one minus the fractional oxygen consumption. of fluorescence is suggestive of a flavoprotein. not sufficiently

precise to distinguish

many flavoproteins

such as a slight

These present results are

other characteristics

red shift

exhibited

by

of the 445 nm peak.

If the reaction products are allowed to decay by standing for 15 minutes or warming to room temperature, the absorption curve C results. is identical exactly

to free FMNwith an optical

to that predicted

Rate of Oxygen Utilization

This curve

density at 445 nm corresponding

from the added FMNHz. and Appearance of FMN

The response time of the oxygen electrode prevented measurementsof the oxygen concentration

being made in time intervals

rate of oxygen utilization

The

is compared in Figure 2 to the appearance of opti-

cal density at 445 nm. The insert tion is in two steps, the first psuedo first

less than one minute.

in figure

2 shows that the oxygen utiliza-

being quite rapid followed by a second slower

order uptake of oxygen with a t4 of 1.3 min.

161

The initial

increase

Vol. 53, No. 1,1973

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AND BIOPHYSICAL

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in OD 445 is followed by a slower increase which corresponds to the long-lived intermediate

decay (t&,7 min) and presumably represents the conversion of

flavoprotein

to free FMN. The first

oxygen utilization

rate probably corres-

ponds to the rapid OD445 appearance which is at a rate far slower than for oxygen oxidation

of FMNH2in free solution.

that the initial

OD 445 production

The preliminary

is not first

results

indicate

order.

Hydrogen Peroxide If oxygen utilization

is measured in the presence of catalase (0.1 mg/ml)

it is found in the first added.

If the reaction

equivalents

minute 0.5 equivalents

of H20z are formed per FMNH2

products are allowed to decay (15 min) another 0.5

of Hz02 are produced. DISCUSSION

These results may be described by the following designate the two intermediates E +

zFM2

as XI and X2.

+ 02

E

Fast”

schemein which we will

The initial

step is

X1 + H202 + FMN

Two moles of FMNH2react with oxygen and luciferase

to yield

one mole of Hz02,

one mole of free FMNand one mole of enzyme bound FMN. This represents an overall

process composedno doubt of a sequence of reactions.

The intermedi-

ate XI then takes up another mole of oxygen at a second slower rate to yield X2 which in the presence of aldehyde, gives rise to bioluminescence or in its absence, decays in a first

order manner with a half-life

(tlr 1.3 min.) Xl

+ 02

of 7 minutes, that is:

(t4 7 min.) >

> E + FMN+ H202

x2

RCHO I E + FMN+ H20 + RCOOH + hv It would appear reasonable that Xl is a flavoprotein Disulfide

bond reduction

to cysteines would fulfill

suggested by the observed inhibition

of the light

162

of composition HzE-FMN. this role (8) and ichis is reaction

by arsenite

(4).

Vol. 53, No. 1,1973

Oxygen addition

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yields Xp which is probably a sulfur hydroperoxide.

hydroperoxide must be eliminated as the absorption spectra respond to fully

oxidized

of Xl and X2 cor-

free and bound FMNand not to flavin

which have maximumabsorptions at 410 or below 380 nm,with little at 445 run (9,lO).

A flavin

hydroperoxides contribution

The composition of X2 must be HOOHE-FMN which explains why

it can break down to Hz02 with release of FMNor react with aldehyde to give bioluminescence. Acknowledgement We thank Dr. Y. H. Li for assistance in the fluorescence lifetime measurement. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

10.

Lee, J., Biochemistry II, 3350 (1972). Lee, J., and Murphy, C., in ~*Chemiluminescenceand Bioluminescence," Cormier, M. J., Hercules, D. M. and Lee, J., Eds., Plenum Press, NewYork, p. 381 (1973). Shimomura, O., Johnson, F. H., Kohama,Y., Proc. Nat. Acad. Sci. (U.S.) 69, 2086 (1972). Hastings, J. W., and Gibson, Q. H., J. Biol. Chem. 238, 2537 (1963). Hastings, J. W., Weber, K., Friedland, J., Eberhard, A., Mitchell, G. W., and Gunsalus, A., Biochemistry 8, 4681 (1969). Wampler, J., and DeSa, R., Applied Spectroscopy 6, 623 (1971). Gibson, Q. H., and Hastings, J. W., Biochem. J. 83, 368 (1962). Hastings, J. W., Riley, R. H., and Massa, J., J. Biol. Chem. 246, 1473 (1965). Spector, T., and Massey, V., J. Biol. Chem. 247, 5632 (1972). Yamasaki, M., and Yamano, T., Biochem. Biophys. Res. Comm.51, 612 (1973).

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