Oxidation-reduction reactions in the photosynthetic bacterium Chromatium. II. Dependence of light reactions on intensity of irradiation and quantum efficiency of cytochrome oxidation

ARCHIVES

OF

BIOCHEMISTRY

AND

Oxidation-Reduction Chromatium.’ Irradiation

the Johnson

Reactions

(1960)

in the

Photosynthetic

Bacterium

II. Dependence of Light Reactions on Intensity and Quantum Efficiency of Cytochrome Oxidation JOHN

Prom

88, 40-53

BIOPHYSICS

Research

M. OLSON2 Foundation,

BRITTON

AND

University Received

of Pennsylvania,

August

of

CHANCE Philadelphia,

Pennsylvania

21, 1959

The intensity dependences of light-induced reactions in Chromatium have been determined spectrophotometrically. The intensities of near-infrared irradiation required for half-maximum oxidation of the various cytochrome species are in the proportion 1:500:~4000 for C4W.6, C4.26, and the combination of C&z% and C4SO. The quantum requirement for intracellular cytochrome .@?.6 oxidation has been determined by measuring the initial rate of absorbancy change (423 mr) in anaerobic bacteria upon irradiation with monochromatic yellow light (589 rnp) of known quantum intensity. This requirement is 2 quanta per electron at both 11 and 29°C. A hypothetical structure for the cytochrome system in Chromatium consists of three distinct electron-transfer pathways. The pathway including C 425.6 is of primary importance in photosynthesis.

ties. By this method Duysens (6) has shown a difference in the response of two cytochromes in Rhodospirillum rubrum to illumination. In the present paper a study of absorbancy changes in Chromatium, strain D, as functions of intensity is described, and marked differences in the response of the various cytochrome species to irradiation are demonstrated. When an intracellular oxidation-reduction reaction is observed upon irradiation of a photosynthetic organism, a general method can be applied for determining whether the reaction is involved in photosynthetic electron transfer or is merely a side reaction. This method is the measurement of the quantum efficiency for the reaction in question (7). A preliminary note (1) reported the quantum efficiency for intracellular oxidation of cytochrome in Chromatium during low-intensity irradiation. In the present paper the quantum efficiency experiments are described in detail, and the participation of one cytochrome as an intermediate in photosynthetic electron transfer is established.

INTRODUCTION

In the preceding paper (2) it has been shown that irradiation of Chromatium with near infrared causes the oxidation of intracellular cytochromes and, under certain conditions, the accumulation of a pigment designated as P 43.2. Similar observations have been made with other photosynthetic bacteria (3-5). Spectral and kinetic analyses of light-induced absorbancy changes have enabled the identification of a number of pigments which react upon illumination. A complementary approach in the investigation of these pigments is the measurement of absorbancy changes over a range of intensii Taken in part from a dissertation presented by John M. Olson to the faculty of the Graduate School of the University of Pennsylvania in partial fulfillment of the requirements for the Ph.D. degree, June 1957. A preliminary note on this subject has been published (1). 2 Predoctoral Fellow of the National Science Foundation. Present address: Graduate Department of Biochemistry, Brandeis University, Waltham 54, Massachusetts. 40

INTENSITY

DEPENDESCE

OF

LIGHT

RESCTIOXS

IN

41

CHROMATIUM

integrating sphere out G\ line j .,.--‘-‘-‘-...~, .c condensing

Osram sodium arc lamp

FIG. 1. Optical setup for illumination of sample with 589-rnr beam in either integrating sphere or spectrophotometer. The effective distance between lamp and sample is about 11 cm., which is twice the focal length of the objective lens. The beam can be turned on and off by means of a spring-loaded hand shutter. METHODS The preparation of the bacteria urement of absorbancy changes beam metshod have been described 1. IRRADIATION

OF

and the measby the doublepreviously (2).

BACTERIA

Samples were irradiated with monochromatic yellow light (X = 589 rnp) by means of the optical setup shown in Fig. 1. The light from an Osram sodium lamp operated on stabilized direct current was filtered through two Wratten 23A filters in order to remove the emission lines at 467,498, and 568 rnp. A Corning 978 filter was also required to remove the considerable near-infrared emitted by the lamp. When samples were irradiated in the a Corning 5443 filter was spectrophotometer, placed between the sample and the photomultiplier to absorb scattered yellow light. The high transmission of this filter in the blue-violet region permitted observation of the reactions under invest,igation. Both the sodium lamp and the tungsten projection lamp previously described (2) were used as sources of near-infrared radiation. Without the Corning 978 filter in place (Fig. I), the actinic effect (on bacteria) of the near-infrared radiation emitted by the sodium lamp was several times the actinic effect of the 589-m@ light. In order to st,udy the effects of very low intensity near-infrared irradiation (see Figs. 3 and 4), so-called “neutral density” glass filters were used to att.enuate the beam. This made the 589-mr component completely negligible compared to the actinic near-

infrared radiation, since the density of each filter at 589 rnp was three times the average density in the region 8ON50 rnp. The tungsten lamp was used to study the effects of high intensity irradiation (see Figs. 4 and 5), as it could provide up to 1400 times the maximum near-infrared intensity available from the sodium lamp. The optical setup shown in Fig. 1 was modified so that the tungsten lamp replaced the sodium lamp, and one Wratten 88A filter replaced those shown. By means of calibrated interference filters (Balzers) and a calibrated thermopile (Eppley), the following values of spectral intensity of irradiation were found at 799, 850, and 908 rnp, respectively: 15, 19, and 16 X lO+ w./sq. cm./mw. The maximum actinic irradiation (I,,,) therefore had an average spectral intensity of about 17 X 10-e w./sq. cm./mp in the region for which the light attenuation filters were calibrated (80&850 mp), In order to obtain a rough comparison with the illumination intensities mentioned by other workers, the near-infrared spectral intensity from a tungsten source operating at an approximate color temperature of 2980°K. was compared to the luminous intensity as measured by a foot-candle meter (Photovolt model 200). The luminous int,ensity corresponding to I,,, was found to be 240 f 30 ft.-candles at t.his color temperature. 2.

DETERMINATIONOFQUANTUM

INTENSITY

The quantum intensity of the 589-rnH striking the sample cuvette at right angles measuring beam in the spectrophotometer

beam to the was

42

OLSON

AND

measured by means of a Cetron CE-31-V photodiode, which had been calibrated by measuring spectrophotometrically the photodissociation of carboxymyoglobin corresponding to a given phototube signal. This method of determining the quantum intensity is a modification of that described by Chance (8) and is based on the quantum efficiency of carboxymyoglobin photodissociation (d, = 1) established by Bticher and Negelein (9) and shown to be essentially independent of wavelength by Biicher and Kaspers (10). (a) Theory The reaction between ferromyoglobin and carbon monoxide is a simple bimolecular reaction. Mb + CO + e-p

MbCO

5

P

At equilibrium the fraction of myoglobin bined with carbon monoxide is given by p/e

= (1 -t kzlka)-1,

com0)

where e is the concentration of ferromyoglobin plus carboxymyoglobin, p is the concentration of carboxymyoglobin, x is the concentration of CO, kl is the rate constant for association, and kz is the rate constant for dissociation. Upon irradiation of the system with monochromatic light, the rate constant for dissociation becomes kz + k’z , where k’, = ,&+1x . ~3~is the molar absorbancy indexa of MbCO, 6 is the number of molecules dissociated by the absorption of one quantum of light, and 1~ is the quantum intensity of the monochromatic light. If the CO concentration is essentially the same in the light and in the dark, the difference in the fraction of MbCO between the irradiated state and dark state is --p/e

= k’dp/e)dark(klx

+

kz

+

VP.

(2)

Upon irradiation, the transition from the dark equilibrium state to the irradiated equilibrium state satisfies the differential equation 1 dp = klx -

ii dt

(k,x

+ kz f

k’z)p/e.

If x is assumed constant, the transition is an exponential of time constant, ro,, =

(klx

+

(3) of p/e

kz + k’$I.

8 Defined by the equation In T = -pcd, where is the transmission of a solution of concentration c and thickness d. T

CHANCE

Similarly the transition from the irradiated equilibrium state back to the dark equilibrium state is an exponential of time constant, 7orr = (klx + kt)-1. If k’z is large enough, the difference between the reciprocal time constants of the light-on and light-off transitions may be used to calculate k’z and I. However, if k’z is less than the precision with which the reciprocal time constants can be measured (as was the case in this study), then it must be calculated using the relative change in the equilibrium value of p upon irradiation [Eq. (2)l. k’z

=

(-AP/Pdsrk)

(lhon)

(4) = (-AP/Plight) (l/roff) The ratio of tb89/~7 for horse MbCO was measured and found to be 0.413. Using the value of t&77 found by Bowen (ll), the value of ebs9 was calculated to be 5.3 m&f-r cm.+, which is in good agreement with the value of 5.4 previously calculated by Chance (8) from data of Theorell and de Duve. The value of p689 is thus 12.3 mM-lcm.-l, the value of 4 is one (9, lo), and values of I are readily obtained from values of kfp . (b) Procedure

A 3 &f solution of dialyzed horse myoglobin in 0.1 M phosphate buffer, pH = 7.0 and t = 2”C., was placed in the double-beam spectrophotometer with the monochromators set to 423 and 470 rnp, respectively. The reduction of the myoglobin by a trace of added Na&&04 was denoted by an increase in absorbancy at 423 rnp. The reduced myoglobin was titrated with microliter aliquots of CO-saturated distilled water. A positive A@423 0410) denoted the formation of MbCO. In a typical case, CO was added until the MbCO concentration reached approximately 2.6 pM. The value of p&k was calculated from the total absorbancy change. For each photodissociation recorded, the value of Ap was calculated from A(0423 - D~&,ax or A(Daa - D4aa)max and the values of l/7,, and 1/7,ff were evaluated graphically by plotting the logarithmof 1 - A(D~z~ - D,,r)lA(Dazs - Dref)msX versus time for both the light-on kinetics and the light-off kinetics. The values of 7 were about 10 sec. The calibration of the phototube was made on the basis of eight experiments. In each experiment the photodissociation of MbCO was recorded about seven times. A value of @+I corresponding to each photodissociation was computed, and the average value was then calculated for the experiment. The proportionality constant was calculated by dividing the sum of the average values of &I by the sum of the corresponding signals from the phototube (Table I). The probable error in the proportionality constant was calculated to be =l=7%.

INTENSITY

DEPENDENCE

OF LIGHT

REACTIONS

Various combinations of “neutral density” filters, microscope slides, and Wratten 22 filters were used to attenuate the 589-rnp beam in experiments with bacteria. These combinations were calibrated at 589 mp by means of a Beckman DU spectrophotometer.

3. ABSOLUTE

ABSORPTION OF BACTERIAL SUSPENSIONS AT 589 rnp

For the determination of the quantum efficiency of cytochrome oxidation, the fraction of 589-rnp light absorbed by each bacterial suspension was measured by means of an integrating sphere (Fig. 2) together with the illuminating setup shown in Fig. 1. The method is similar to those used by Kok (12) and Rieke (13) for measuring the absorption of algal suspensions. The beam of 589.mp light from the sodium-arc lamp enters the sphere through the front entrance port. The 8 mm. X 30 mm. image of the stop shown in Fig. 1 is focused near the center of the sphere so that the entire beam strikes either the sample cuvette or the opal glass. In an ideal sphere all the light reflected or scattered from either the sample or the opal glass contributes to the average illumination of the sphere independently of the scat,tering pattern. The light flux passing out, of the exit port strikes a photomultiplier (Fig. 2). The electrical signal, which is proportional to the total amount of light leaving the sample or the opal glass, is compared to a reference voltage, and the difference is amplified and recorded by the electronic equipment associated with the doublebeam spectrophotometer (2, 14). The mask between the center of the sphere and the exit port prevents any light reflected or scattered from the sample from striking the photomultiplier without first being diffusely reflected by the walls of the sphere. The opal glass provides a TABLE CALIBRATION

Phototube sec.-’

,003o .0033 0025 .0025 .OOli .0020 .0013 .0016 __ Zmv

=

(1.38

I

OF PHOTOTUBE

f

signal

AT

589 rng

Proportionality constant

mz’.

10-r sec.7 mr.-1

20.8 20.6 19.6 19.4 12.8 12.6 12.1 11.6

1.4 1.6 1.3 1.3 1.3 1.6 1.0 1.3

JO) X 10m4 sec.?

mv.-’

IN CHROMATIUM

43

photomultiplier

lever arm c, c==~~=~~z

FIG. 2. Integrating sphere: rear-view cross section. The sphere is made from two copper hemispheres; the inside diameter is 9 in. The front hemisphere has a rectangular entrance port, 5 mm. x 15 mm., in the front and a circular exit port 49 in. in diameter on top. The inner surface of the sphere and all the metal fixtures are painted with white enamel which is covered with a layer of barium sulfate. The positions of the flash opal glass and the cuvette can be interchanged by means of the external lever. standard to which each sample is compared (13). The apparent transmission (t) of any material in the cuvette is defined as the ratio of the signal when the cuvette is in the light beam to the signal when the opal glass is in the beam. This apparent transmission is independent of the intensity of the beam and is also independent of the efficiency of the sphere. This method eliminates transmission reading variations caused by slow changes in the brightness of the source or by opening and closing the sphere. Furthermore, the apparent transmission should be essentially independent of the reabsorption by the sample of light reflected from the walls of the sphere, since the sample remains inside the sphere u-hen the opal glass is in the beam. In practice the t)rue absorption (A = 1 - 2’) of a bacterial sample is calculated from the apparent absorption (a = 1 - t). In this calculation, corrections must be made for two sources of error: Some light is reflected from the front surface of the

44

OLSON AND CHANCE

cuvette without entering the sample, and the sphere does not integrate the light scattered from the sample completely independently of the turbidity of the sample. The first correction is given by the apparent absorption of India ink in the cuvette (approximately 90%). The second correction is given by the apparent absorption of a nonabsorbing substance, e.g., diluted milk, having the same turbidity as the sample. A convenient index of sample turbidity is 1 RIOOO , where RMCMis the reading at 1000 mp on the transmission scale of a Beckman DU spectrophotometer. Since the apparent absorption of milk or magnesium oxide suspensions as measured at 589 rnp in the integrating sphere is a linear function of 1 - Rlooo , the correction for turbidity is easily obtained from the value of RIOOO for a given sample. The true absorption of a sample is given by the following equation derived elsewhere (14) :

relatively constant at a value of 5 sec. Also, the transitions to the irradiated steady state and back to the dark steady state were approximately exponential with respect to time. However, as the intensity was increased beyond 2 X lop4 I,, , the initial change upon irradiation was too fast for an exponential transition, and the initial change after irradiation was too slow for an exponential return to the dark steady state. In this intensity range of non-exponential kinetics, the half-time for the return to the dark steady state increased steadily and with increasing intensity, approaching the value characteristic of the third (slow) phase of the light-off transition in the anaerobic light effect at high intensity (2). This increase in half-time showed that the steady-state electron flux to primary oxidant A = bsamplo - hilk)/(aink - &ilk) via cytochrome 423.5 did not increase in proThis method for measuring the absorption of portion to the intensity of irradiation when bacterial suspensions has an accuracy of at least the intensity exceeded 2 X 10e4 I,,,, . In two significant figures for values of absorption in fact, the slow reduction of cytochrome upon the range of 20-30’%. cessation of irradiation at these higher int’ensities suggesteda general accumulation of RESULTS oxidized material during the light interval and a limitation in the rate of electron trans1. IRRADIATION OF BACTERIA WITH fer from substrate. The steady-state absorbNEAR INFRARED ancy difference at 422 rnp reached a plateau (CL)Changes in the Anaerobic Light h’j’ect at about 10e3 I,,, . A tenfold intensity inwith Intensity creaseto 1OV I,,, increased the absorbancy The light effect at low intensity (I < 10m3 change only 1.5%. Presumably C 423.6 was I,,,) showed important differences from the 100 % oxidized in the irradiated steady state light effect at high intensity (I,,,) described at these intensities. As the intensity of near-infrared irradiapreviously (2). In contrast to the complexity tion was increased to about 2 X 10e2I,, , of the polyphasic kinetics characteristic of the characteristics of the anaerobic light efhigh intensity irradiation, both light-on and light-off transitions in absorbancy were mono- fect began to change markedly (see Fig. 4). The curve of the st’eady-stat’e absorbancy phasic at low intensities of near-infrared irradiation, and indicated the reaction of difference versus logarithm of irradiation inonly one cytochrome. The spectrum of the tensity began to curve upward once again, total absorbancy change produced by ir- and the light-on and light-off transitions beradiation at 10e3 I,,, had a trough at came diphasic. The separation of the lightoff transition into two distinct phases was 423.5 rnp and showed t,hat this cytochrome especially clear-cut. These two phases corwas C 423.5 (2). In Fig. 3 the steady-state absorbancy responded to the intermediate (second) and change at 422 rng and the half-time of the slow (third) phasesof the light-off transition light-off transition are plotted versus in- in the anaerobic light effect obtained at maximum intensity (2). As the intensity of irtensity over a range of low intensities (I < 10+1,,). Up to an intensity of 1.5 X 1O-4 radiation increased from 2 X 10e2 Imax’to I max the steady-state change was propor10-l Imax ) the increase in the steady-state tional to intensity, and the half-time was absorbancy change was accompanied by a

INTENSITY

DEPENDENCE

OF

LIGHT

REEACTlONS

IN

CHROMATIUM

-.006 -40

a

0

00

I

2

3

4

6

5

7x10-4

I/I max 3. Variation in two characteristics of the anaerobic light effect caused by low intensity near infrared. Curve A is the steady-state absorbancy change at 423 rnp. Curve B is the half-time for the transition back to the dark steady state. FIG.

corresponding increase in the magnitude of the faster phase of the light-off transition. The slow phase remained essentially the same, independent of intensity. As the intensit’y was increased beyond 10-l I,,,, ) the light-off transit’ion became distinctly triphasic. The t’hree phases corresponded to those of the off reaction in the anaerobic light effect obtained at maximum intensity (2). Above 2 X 10-l I,,, , the magnitude of the intermediate phase of absorbaney change was constant. At I,,, the magnitude of the fast phase had almost reached a plateau also. When the magnitude of the absorbancy change in either the intermediate phase or the fast phase of t’he light-off transition was plotted versus “intensity” instead of “logarithm of intensity”, as in Fig. 4, a linear extrapolation to the origin could be made from the data. This suggestedthat the lightsaturation curves for these two phases may also begin at the origin as linear functions of int,ensity as does the saturation curve for the slow phase (see Fig. 3). If this be the case, the slopes at’ the origin for the slow, intermediate, and fast phasesare in the approxi-

.03-

-0

.02:

:

IY 3 .Ol-

.oo -5

-4

-3

-2

-I

0

LOPI/Inlax 4. Anaerobic light effect versus logarithm of intensity. Curve A is the total absorbancy change at 422 rnp caused by near infrared. Curve B is the magnitude of the slow phase of the light-off transition. Curve C is for the intermediate phase, and Curve D is for the fast phase. FIG.

mate ratio 1000: 5: 1. The sharply defined differences in intensity requirement for the reactions reflected in the three phasesof the light-off transition are also shown by the intensities required for half-maximum absorbancy change in each case (seeTable II).

46

OLSON AND CHANCE

TABLE II RELATION OF REACTION RATES AND STEADY STATES TO INTENSITY OF NEAR-INFRARED IRRADIATION R,, is the initial rate of absorbancy decrease at 422 rnp upon irradiation. k,rr is the ratio of the initial rate of absorbancy change (light-off transition) to the steady-state absorbancy difference. 11~ is the intensity of near infrared required for the half-maximum steady-state absorbancy difference. Light effect Anaerobic light effect (I < lo-47,,,) (I < lo-ZZ,,,) (I > lo-2Zmax) Slow phase Intermediate phase Fast phase Aerobic light effect

Ron X Imex/I sec.-'

korr sec.-1

IlldIrnex

1.4 x 10’ 1.4 x 10’

1.6 X 10-r Variable

1.3 x 10-4

1.4 X 10-z

2 x 10-a 10-t 5 x 10-r 5 x 10-l

6 x 1O-2 3-7 x 10-i 3-7 x 10-r

absorbancy change apparently became faster than the response of the measuring apThe light-induced absorbancy change at paratus. In aerobic bacteria the initial rate of ab422 rnp in aerobic bacteria became detectable only when the intensity of irradiation ex- sorbancy change at 422 rnp upon irradiation ceeded 1O-2 I,,, . The dependence of the (interpreted as oxidation of C 422) was also aerobic light effect on intensity was essen- found to be proportional to intensity, but tially the sameas the dependence of the fast the intensity required for a given rate of abphase of the light-off transition under ana- sorbancy change was approximately 1000 erobic conditions. The absorbancy change in- times the intensity required under anaerobic creased in proportion to the intensity for conditions. weak irradiation, but gradually leveled off to 2. QUANTUM REQUIREMENT FOR a constant maximum value as the intensity CYTOCHROME OXIDATION approached I,,, . The quantum efficiency of C 423.5 oxidation in anaerobic bacteria was determined (c) Initial Rate o.f Absorbancy Change upon by measuring initial rates of cytochrome Irradiation oxidation upon irradiation with known inThe spectrum of the initial rate of absorb- tensities of monochromatic light. Since very ancy change in the Soret region was de- low intensities were used for irradiation, the termined for irradiation of anaerobic bacteria oxidation of other cytochrome species was at a given low intensity (4 X lop4 I,,,) be- completely negligible. A sodium lamp was low saturation of the slow phase of the lightused as a source of monochromatic radiation off transition. The maximum rate (approxibecausethe dominant 589-rnp emission band mately - 0.005 sec.-l in a typical sample) of sodium is so close to the 590-m,u absorpoccurred between 422 and 423 rnb, and the tion peak of bacteriochlorophyll in Chromaspectrum was similar to that of the total ab- tium. Since the 590-rnp peak is relatively sorbancy change caused by irradiation at small in comparison to the peaks in the near low intensity. infrared, suspensions taken directly from The initial rate of absorbancy change at growing cultures and placed in 1 cm. X 1 422 rnp was found to be proportional to in- cm. cuvett.es absorbed lessthan 30 % of the tensity up to about 2 X lo-* I,,, . Above 589-m@light. This assuredthat all parts of a this intensity value, the curve of initial rate given sample were exposed to intensities of versus intensity began to bend toward the the same order of magnitude. The equation for the average rate of intensity axis. At high intensities. the initial (b) Ch.angesin the Aerobic Light E$ect with Intensity

INTENSITY

DEPENDENCE

OF

LIGHT

REACTIONS

f7h.J

IN

47

CHROMATIUM

(mg/sec.)

FIG. 5. Initial rate of absorbancy change at 423 rnp in anaerobic cells versus rate of 589-rnp light abso&ion. The sloDe of Curve A is 62 cm.-’ mE-1 and the slope of Curve B is 40 cm.-1 mF1. The data are f&m Expt. 4 oiTable III.

589-rnp light absorption (millieinsteins/l./ sec.) in a sample is

Rhv = A,TJb

X illuminated area of sample/vol. of sample

where A, is the absorption of the sample (obtained by means of the integrating sphere), Tf is the transmission of the filter combination used to attenuate the actinic beam, and Ib is the quantum intensity of the unattenuated beam (mE cm.-2 sec.?). Since one side (1 cm. X 3 cm.) of the sample (1 cm. X 1 cm. X 3 cm.) was uniformly illuminated,

Rhv = A,TJb

cm-‘.

Initial rates of absorbancy change at 422423 rnp upon irradiation of anaerobic samples were measured. Each transition was recorded by the photographic oscillograph, and the initial slope was taken from the “best” tangent to the initial portion of the light-on transition. Each sample was irradiated at several intensities: 10 %, 20 %, etc., up to the maximum (100 %) intensity available from the optical setup shown in Fig. 1. This setup was used both with and without the stop in place next to the condensing lens. Without the stop the intensity at the sample was about 70% greater than with the stop in place. The maximum intensity of monochromatic 589-rnk light caused about the

same absorbancy change as did near-infrared radiation of intensity 2-3 X 1O-4I,,, . The results of a typical experiment are shown in Fig. 5, where the initial rate of absorbancy change at 423 rnp is plotted versus Rhv . The scatter in the data reflected both the variability in the responseof the bacteria to irradiation and the imprecision in measuring the initial rates. Nevertheless, the evidence clearly demonstrated that the initial rate of cytochrome oxidation is proportional to radiation intensity throughout the range observed. The range of variability in the response of the bacteria to irradiation is shown by lines A and B in Fig. 5. In each sample studied the limits of variat.ion from the average slope, -d (0422 - D470)/dt versus Rhv , were f20-30 %. The detailed results of all experiments are given in Table III. The average results (neglecting Expts. 1 and 2) may be summarized in the following equation:

[d(Daw -

D4TO)ldtlinitial

=

-KRhv

,

where K ‘v 50 cm.-’ mE-1. For b-type and c-type cytochromes generally, Ae W 20 mM+ cm.-’ at the a! peak in the reduced-minus-oxidized difference spectrum (15). The difference spectrum of C 423.5 has similarities to both b- and c-type spectra. Therefore AE~ was assumed to be about 20 mM-’ cm.-l and Ae, was calculated to be about 100 mM-’ cm.-l, since A+./

48

OLSON

TABLE

AND

III

SUMMARY OF EXPERIMENTS ON THE RELATIOXSHIP BETWEEN INITIAL RATE OF CYTOCHROME OXIDATION AND RATE OF ABSORPTION OF 589rnM LIGHT A different culture was used for each experiment. In Expts. 1 and 2 the bacterial concentration in each sample was twice the concentration in the source culture. All experiments were performed at room temperature (-25°C.) unless otherwise noted. Light intensity was varied from 0 to 2 X lo-’ m.?? cm.? sec.?. Key: (a) experiment number, (b) sample medium, (c) number of initial rates measured, (d) fraction of actinic light absorbed (A,), (e) slope cl of -(Dn2~.3 - DUE) versus RnV expressed in dt cm.-’ mE-1. a

b

1. 2.

Culture liquid Culture liquid Fresh medium Average (I and 2) 3. 4. 5. 6.

7.

Culture liquid Fresh medium Culture liquid Fresh medium Fresh medium Fresh medium (29°C.) Fresh medium (11°C.) Fresh medium

Average

c

d

e

15 15 45

.44 .46 .49

24 f 24 f 26 f 25

7 7 6

72 69 56 56 49 28

.21 .21 .26 .27 .22 .24

64 42 51 46 47 52

17 11 11 10 9 13

28

.24

50 f

.078 .14 .21*

37 46 37 50 50 53 62 56 46 49

5 6 6 6 6 6 6 6 6

.28 .35 .40 .54 .63

(3-7)

* Same

f f f f f f

(l/K’) is therefore approximately 2 quanta per electron. The first two experiments (Nos. 1 and 2 in Table III) originally indicated a quantum requirement of 4, but the values indicated by all subsequent experiments lay between 1.6 and 2.7. In Expts. 1 and 2, the bacterial concentration was about twice the concentration used in Expts. 3-6. Experiment 7 was performed to test for a possible dependence of the apparent quantum requirement on bacterial concentration. No significant variation was found as shown by Fig. 6. Therefore bhe results of the first two experiment’s were considered to be inaccurate. In some samples, the bacteria were suspended in culture liquid from which the sulfide had been removed previously during growth of the cult,ure. In other samples, the bacteria were suspended in fresh medium. The results were essentially the same for both types of medium. Experiment 6 indicated that the initial rate of cytochrome oxidation upon irradiation is independent of temperature over the range 11-29°C. In contrast, the rate of cytochrome reduction upon cessation of irradiation was markedlv affected bv temperature. at The half-time of “t’he light-off transition

7

0, 2 a

sample.

LIz 5 [see Table I in preceding paper (2)]. If A~422 - Aedr0 is taken to be 100 m&P1 cm.-‘, the initial rate of cytochrome oxidation is then given by

Ae

f [Fe3+ cyt.1 ) where

CHAiYCE

the quantum

initial

= K’Rhv)

efficiency

M/E. The average quantum

K' is -0.5 requirement

0

.I

.2

.3

.4

.5

.6

WET CELL VOLUME SUSPENStON VOLUME(O/O) FIG. 6. Apparent quantum requirement of cytochrome oxidation as a function of cell concentration. Open circles denote the quantum requirement values calculated from the data of Expt. 7 in Table III. Solid circles show the fraction of 589rnp light absorbed (A& as determined by the integrating sphere method.

INTENSITY

DEPENDENCE

OF LIGHT

11°C. was 16 sec. compared to a half-time of 4 sec. at 29°C. From this experiment it was clear that the measurement of initial rates of oxidation in the light-on transitions was not affected by the higher cytochrome reductase activity at 29°C. A preliminary experiment with Rsp. rubrum indicated that the initial rate of cyt’ochrome oxidation in this organism was of the same order of magnitude as in Chromatium. However, the relatively small total change in Rsp. rubrum upon irradiation made estimates of init~ial rates very crude. Further investigation by one of us (B.C.) is in progress. 3. IRRADIATION

OF AEROBIC BACTERIA OF PHENYLMERCURIC ACETATE

IN

REACTIONS

0

49

IN CHROMATIUM

40

80

120

Imax/

FIG. 7. Aerobic light effect in the presence of 30 PM PMA. The reciprocal of steady-state change in absorbancy at 436 rnp is plotted versus reciprocal intensity of near-infrared.

chromes are grouped funct,ionally in three different electron-transfer systems, each of which is capable of donating electrons to the The absorbancy increase at 436 rnp upon primary oxidant(s) produced photocheminear-infrared irradiation of an aerobic sam- tally. For simplicity it will be assumedthat ple containing 30 1LMphenylmercuric acetate one primary oxidant may accept electrons (PMA) was measured at seven intensities from all three cytochrome groupings. This from 1OWI,,, to I,,, . [This absorbancy in- interpretation is pictured in Fig. 8. crease has been attributed to the accumulaThe cytochrome which reacts most rapidly tion of a pigment P @S (2).] The data fit the upon irradiation of anaerobic bacteria has rectangular hyperbola : been designated C 42S.5 (2). This cytochrome is responsible for the slow phase of .0042 the light-off transition after high-intensity A(o436 - 0470) = 1 + .0336 I,,,/I irradiation. C 42S.5 may be pictured as the This is shown in Fig. 7, where the reciprocal terminal member of one electron-transfer absorbancy increase is plotted versus re- pathway (I) and as that cytochrome which reacts most rapidly with the primary oxiciprocal intensity. The intensity requirement for this light effect may be compared to the dant. After irradiation at very low intensirequirements for the other light effects on ties (I < lop4 I,,,), the rate of reduction of C 42S.5 (indicated by the speed of the lightTable II. off transition) is relatively high. However, DISCUSSION this rate of reduction actually decreaseswith increasing intensity of irradiation up to an 1. STRUCTURE OF CYTOCHROME SYSTEM intensity of about 2 X lop2 I,,, , where the Some general characteristics of the cyto- rate of reduction is only -1 or 2 % of the chrome system in Chromatium have been rate after low-intensit’y irradiation. The reapresented in the preceding paper (2). A more son for t’his phenomenon might be a decrease detailed concept of the cytochrome system in C 42S.5 reductase activity with increasrequires an interpretation which, although ing intensity of irradiation and/or an acnot unique, is qualitatively consistent with cumulation of oxidant which persists after the data. The following proposed mechanism cessation of irradiation. Since the addition of may be useful in correlating the experimental colloidal sulfur to a bacterial suspensioninobservations. creasesthe rate of C 429.5 reduction in the On the basis of the three distinct phases slow phase of the light-off kansition, pathof the light-off transition in the anaerobic way I apparently t’ransfers electrons from light effect (2), it is postulated that the cyto- exogenous inorganic substrates. THE PRESENCE

50

OLSON

AND

CHANCE

[CO, fixation system] 2 hv

+

bacterioP chlorophyll

C422+

+

C430 +.-e(PATHWAY III)

reductant 2

/ XY

“Y”
*\I J/

C423.5 +*--dedehydrogenases+ (PATHWAY I) PMA .~- dehydrogenasese C426 4 \ (FdTHWAY II) 60,CN’

(?I inorganic S compounds (?I J substrates

02 FIG. 8. Hypothetical mechanism of electron transport in Chromatium. The arrows indicate direction of electron transfer. The dotted sections denote a series of unknown reactions. The dashed lines indicate inhibition of electron transfer.

The CO-binding cytochrome C 426 is responsible for the intermediate phase of the light-off transition under anaerobic conditions. This cytochrome is conceived as the terminal member of a second electron-transfer pathway (II). C 426 is not involved in the first phase of the light-on transition and, compared to C 423.6, requires about 500 times higher intensity for a half-maximal steady-state change upon irradiation. Since the steady-state concentration of oxidized C 423.5 becomes independent of intensity before the change in steady state of C 426 with intensity becomes measurable, C 426 is thought to react directly with a primary oxidant at a much lower rate than does C

423.6. Cytochromes C 422 and C 430 have been shown to be closely linked in the aerobic light effect and, by inference, in the first phase of the light-off transition under anaerobic conditions. These cytochromes are placed together in a third electron-transfer pathway (III). C 422 is oxidized more rapidly than C 430 upon irradiation of aerobic bacteria, and is therefore considered to react directly with a primary oxidant, but at a rate some 1000 times less than that of C 426.5. C 430 is thought to transfer electrons to C 422. The rate of reduction of the cytochromes in pathway III upon cessation of irradiation is very high compared to the rates in path-

ways I and II after high-intensity irradiation, but is of the same order of magnitude as the rate in pathway I after very low-intensity irradiation (I < low4I,,,). The light-on transition in the anaerobic light effect at high intensity showsa distinctive plateau between the first and second phase (2). The plateau suggeststhat the oxidation of C 426 and C 422 at an observable rate requires a rather high concentration of primary oxidant which is not available until the rate of electron transfer in pathway I drops from its original high value (shown in the light-off transition after low-intensity irradiation) to its much lower steady-state value. The kinetics of C 422 oxidation in the aerobic light effect show no observable induction period, since there is almost no reduced C 423.5 nor C 426 available to retard the rapid buildup of primary oxidant concentration. In the presence of oxygen in the dark, C 426 and C 423.5 are oxidized, whereas C 422 and C 430 are reduced. The CO-binding C 426 is thought to react directly with molecular oxygen, and then to react with C 423.5, thus providing a link between pathways I and II. Cytochromes 422 and 430 in pathway III

are thought

to be isolated

from

C

426 and C 423.5 and the influence of oxygen. This hypothetical scheme (summarized in Fig. 8) does not attempt to explain the rela-

INTENSITY

DEPENDENCE

OF

LIGHT

tionship between the oxidation-reduction reactions in the cytochrome system and the reactions involving P 43.2. Even in the absence of PMA, the accumulation of P 432 upon irradiation occurs to a small extent under both aerobic and anaerobic conditions. Under aerobic conditions, this accumulation becomes pronounced when cytochrome reductase activity is very low due to either old age of the bacterial culture or inhibition by PMA.

REACTIONS

IN

CHROMATIUM

51

tophosphorylation in particle extracts of Chromatium. Newton and Kamen found that the rate of light-induced formation of ATP reaches saturation at ~300 ft.-candles illumination with white light from a tungsten source. Similarly, Fuller and Anderson observed saturation between 200 and 400 ft.candles. The half-maximal rate of phosphorylation took place at roughly 150 ft.candles (17). The intensity of near infrared required for half-maximal steady-state change in cytochromes 422 and 430 in path2. CORRELATIONS WITH PHOTOSYNTHESIS way III is about 0.3-0.7 I,,, which is equivAND PHOTOPHOSPHORYLATION alent to 70-170 ft.-candles illumination from a Wassink and co-workers (16) have meas- tungsten sourceof color temperature 2980” K. ured COz uptake in Chromatium as a func- (see Methods: 1. Irradiation of Bacteria). tion of light intensity for a number of physi- Anderson and Fuller further determined that ological conditions. At 29°C. the rate of COz the rate of inorganic phosphat,euptake in the uptake was found to be a linear function of presence of a hexokinase trapping system the intensity of 589-rnp irradiation over the for ATP is about 2 X 1OV’ PM inorganic range of 0 to ~2 X lo3 ergs/sq. cm./sec. for phosphate/sec./mg bacteriochlorophyll wit.h bacteria suspended in a medium containing illumination of 300 ft.-candles. With reason1% H&O3 at pH 7.6, and gassedwit,h 5 % able assumptions of the bacteriochlorophyll CO, . (Under the most favorable conditions content of whole cells and t,he absorbancy of CO2 concentration and substrat,e concen- index of C 422, the initial rat.e of C 422 oxitration, the linear region of CO2 uptake ex- dation upon irradiation of aerobic bacteria tended ashigh as 1.5 X lo4 ergs/sq. cm./sec.) may be calculated tjo be 4 X lo-* M&! cytoIn the present study, the CO, partial pres- chrome/sec./mg. bact,eriochlorophyll at an sure could not exceed 0.02 atm. and the intensity 1.2 I,,, (near-infrared intensity H&03 concentration was less than 0.1%. equivalent to 300 ft.-candles). Although there Therefore, under the conditions of this is no direct evidence for t’he coupling of phosstudy, phot’osynthesis as measured by CO2 phorylation to electron transfer in pathway uptake would not be expected to be a linear III, the rough correlations between intensity function of light intensity above 2 X lo3 requirements and reaction rates for C @L?? in ergs/sq. cm./sec. This upper limit to the linear vivo and photophosphorylation in vitro sugregion for 589-my radiation is roughly equiv- gest this possibility. Recent work of Smith alent to a near-infrared int.ensity of 10m2 and Baltscheffsky (19) indicates that photoI max. (This equivalence of 589 rnp and near- phosphorylation may be coupled to electron infrared radiation is based on the intensities transport via cytochrome cg in particle exof each type of radiation required for a given tracts of Rsp. rubrum. initial rat’e of cytochrome oxidation upon irThe suggested coupling of photophosradiation.) Since cytochrome 423.5 in path- phorylation t’o electron transfer via C .@?2 way I is the only cytochrome which shows a raises some questions about the mechanism det’ect,ible responseto irradiation at intensi- of COz fixation and growth. The high light ties below lo-” I,,, , this cytochrome is by intensities required for (7 422 oxidation inimplication the only speciesof importance in dicate a low quant’um efficiency of the order photosynthet’ic electron transport. The very of 0.1 %, and a correspondingly low yield of high intensit,y requirementIs of the oxidation high-energy phosphat.esvia pathway III. In of t,he other cyt,ochromes in pathways II and contrast, COI?fixat,ion, which presumably reIII makes their relevance t,o photosynthesis quires high-energy phosphates in addition somewhat questionable. to a photoreductant, takes place at an effiBoth Newton and Kamen (17) and An- ciency of 1 CO2 molecule/l2 quanta (16). derson and Fuller (18) have measured pho- Furthermore, the curve of CO2 uptake devi-

52

OLSON AND CHANCE

ates from linearity with respect to intensity before the oxidation of C 4.29 is detectable. It would seem, therefore, that whatever phosphorylation is required for COZ fixation must be carried on independently of C 42.2and pathway III. The photophosphorylation of particulate systems which is correlated with C Q?Z, may represent an additional, low-efficiency mechanism for the storage of energy during high-intensity irradiation.

ard free-energy change (AFO) for the net reaction, Fe* cyt + H+ ---f Fe3+ cyt + >sH,,

since AF” is only - .15 kcal. for the reaction of Hz with CO2 to form carbohydrate. The reduction potential for C 42S.5 at pH 7.0 may be considered to be -0.04 v. on the basis of the reduction potential found by Newton and Kamen (21) for Chromatium cytochrome. Since the reduction potential for hydrogen is -0.41 v. at pH 7.0, the value of 3. QUANTUM REQUIREMENTS AF” for the cytochrome reaction is 8.5 kcal. The quantum requirement for C 4ZS.6 oxiSince electronic excitation energy must dation appears to be 2 quanta/electron. This be transferred to bacteriochlorophyll 890 value is qualified by the probable error of in order to be utilized for photosynthesis =t7 % in the determination of light intensity (22), the maximum energy available for and the &30% variation in the observed chemical work is only 32 kcal./einstein. If responseof the bacteria to irradiation. How- the mechanism of photosynthesis in Chroever, the order of magnitude of the quantum matium requires the absorption of 2 quanta/ requirement indicates that C 42S.5 is the electron transferred, the efficiency of energy important intermediate in the t)ransfer of conversion from bacteriochlorophyll in the electrons to the photooxidant produced lowest electronic excitation state (32 kcal./ when Chromntium is irradiat.ed within the mole) to the oxidation of C &B’.S and the range of intensities studied. simultaneous production of a reducing agent The value of 2 quanta/electron agrees is estimated to be about 13 %. If pyridine reasonably well with the value of 12 quanta/ nucleotide is an intermediate in the transfer COZ molecule found by Wassink, Katz, and of electrons to CO2 , the efficiency of energy Dorrestein (16) from experiments in which conversion in the net reaction, the amount of CO2 uptake was measured as a function of the amount of 589-rnp light Fez+ cyt + >$PN+ + >iH+ -+ absorbed. Since about 4 electrons are Fe3+ cyt + >sPXH, required to reduce CO2 to the level of Chrom&urn cell material, 12 quanta/Con mole- is only about 10 %. cule is roughly equivalent to 3 quanta/elecSince the initial rate of C 42S.5 oxidation tron transferred to CO2 . upon irradiation is essentially independent Recently Duysens and Amesz (20) investiof temperature over a considerable range gated the quantum requirement for pyridine extending down to -170°C. (23), a suggesnucleotide reduction by using a fluoro- tion of Kamen (24) is of interest. Kamen metric method analogous to the spectropho- proposes that the mechanism of cytochrome tometric method used for cytochrome oxi- oxidation may involve a direct charge transdation. In Ch.romatium this requirement was fer between chlorophyll in an excited state found to be between 2.8 and 11.2 quanta for and cytochrome. According to this view, a each pyridine nucleotide molecule reduced, relatively small proportion of chlorophyll corresponding to 1.4-5.6 quanta/electron. molecules intimately associated with cyto(The uncertainty in the estimated quantum chrome heme groups serve as sinks for the requirement reflected an uncertainty in the electronic excitation energy being transfluorescence yield of intracellular pyridine ferred by inductive resonance from one nucleotide.) chlorophyll to another. The energy required to oxidize C 42S.5 and ACKNOWLEDGMENTS simultaneously to produce a reducing agent Dr. Paul Latimer made several helpful suggescapable of converting COZ to carbohydrate the integrating sphere. Mr. may be estimated by calculating the stand- tions concerning

INTENSITY

DEPENDENCE

OF

LIGHT

Victor Legallais constructed both the integrating sphere and accompanying optical setup. Dr. Lionel Jaffe and Dr. Jerome Schiff kindly gave us the use of a thermopile and a standard lamp for the determination of radiant intensities. This paper was prepared while one of us (J.M.O.) was a Public Health Service Research Fellow at the Biophysical Laboratory of the National University at Leiden, the Netherlands, and subsequently a member of the Graduate Department of Biochemistry at Brandeis University, Waltham, Mass. REFERENCES 1. OLSON, J. M., AND CHANCE, B., Biochim. et Biophys. Acta 28, 227 (1958). 2. OLSON, J. IX, AND CHANCE, B., Arch. Biochem. Biophys. 88, 26 (1960). 3. DUYSENS, L. N. M., Nature 173, 692 (1954). 4. CHANCE, B., AND SMITH, I,., Nature 175, 803 (1955). 5. SMITH, L., AND RAMIREZ, J., Arch. Biochem. Biophys. 79, 233 (1959). 6. DUYSENS, L. N. M., in “Research in Photosynthesis” (H. Gaffron, ed.), p. 164. Interscience Publ., New York, 1957. 7. CHANCE, B., Discussions Faraday Sot. No. 20, 205 (1955). 8. CHANCE, B., J. Biol. Chem. 202,407 (1953). 9. BUTCHER, T., AND NEGELEIN, E., Biochem. Z. 311, 163 (1942). 10. BUTCHER, T., AND KASPERS, J., Naturwissenschaften 33,93 (1946).

REACTIOSS

IN

CHROMATIUM

5.3

11. BOWEN, W. J., J. Biol. Chem. 179, 235 (1949). 12. KOB, B., Enzymologia 13, 1 (1948). 13. RIEHE, F. F., in “Photosynthesis in Plants” (J. Franck and W. E. Loomis, eds.), p. 251. Iowa State College Press, Ames, 1949. 14. OLSON, J. M., Dissertation, Univ. Pennsylvania, Philadelphia, Pa., 1957 (University Microfilms, Ann Arbor, Michigan.) 15. CHANCE, B., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 4, p. 273. Academic Press, New York, 1957. 16. WASSINH, E. C., KATZ, E., AND DORRESTEIN, R., Enzymologia 10, 269 (1942). 17. NEWTON, J. W., AND KAMEN, M. D., Biochim. et Biophys. Acta 26, 462 (1957). 18. ANDERSON, I. C., ASD FULLER, R. C., Arch. Biochem. Biophys. 76, 168 (1958). 19. SMITH, L., AND BALTSCHEFFSKY, &I., J. Biol. Chem. 234, 1575 (1959). 20. DUYSENS, L. N. hl., AND AMESZ, J., Plant Physiol. 34, 210 (1959). 21. NEWTON, J. W., AND KAMEN, hl. D., Biochim. et Biophys. Acta 21, 71 (1956). 22. DUYSENS, L. N. M., Dissertation, Rijksuniversiteit te Utrecht , The Netherlands, 1952. 23. CHANCE, B., -END ~‘ISHIMURA, M., Proc. Natl. Acad. Sci. U. S. 46, 19 (1960). 24. KAMEN, M. D., Proc. Symposium on Comp. Biol. No. 1. (M. B. Allen, ed.), Kaiser Research Foundation, in press. Academic Press, New York, 1960.