The quantum efficiency for the photochemical conversion of the purple membrane protein.

The quantum efficiency for the formation of M(412), an intermediate product in the photoconversion of the purple membrane protein of Halobacterium halobium, was determined to be 0.30 +/- 0.03 at -40 degrees C. This photochemical reaction was photoreversible to the original pigment and the ratio of the quantum efficiencies gamma PM(568 leads to M(412)/gamma M(412) leads to PM(568) was 0.39 +/- 0.02. No change was seen in either value when exciton interaction between chromophores was eliminated. The sum of gamma PM(568) leads to M(412) plus gamma M(412) leads to PM(568) was 1.07 +/- 0.10, approximately 1, suggesting that the pigment and its primary photoproduct share a common excited state.     

紫膜的紫外荧光及分子内的能量转移

以可见光为作用光照射天然紫膜,紫膜蛋白被280nm紫外光激发所发射的荧光强度比对照略有降低.比较天然紫膜、漂白紫膜与菌蛋白三者的紫外荧光强度,前两者无显著变化,但菌蛋白的荧光强度比天然紫膜的荧光强度大2-3倍,表明生色团对蛋白质荧光可能有猝灭作用.用280nm波长光照射紫膜的暗适应形式,可使其转变成光适应形式.若有羟胺存在,以紫外光照射也可使紫膜漂白.光漂白的作用光谱,其紫外部分与紫膜蛋白部分的吸收光谱重合得很好.上述实验证明紫膜蛋白部分吸收的能量可以转移到生色团上,即紫膜存在分子内的能量转移     

The N intermediate of bacteriorhodopsin at low temperatures: stabilization and photoconversion

In aqueous suspensions of purple membranes (pH 10.2, 0.4 M KCl) an intermediate having an absorption maximum at 570-575 nm (at -196 degrees C) was produced by first heating the M intermediate up to -30 degrees C and then stabilizing it by subsequent cooling to -60 degrees C. We suggest that this species is the intermediate N (or P or R) found and characterized earlier near room temperature. Upon illumination at -196 degrees C N is transformed into a bathochromically absorbing species KN which has an absorption maximum near 605 nm and an extinction 1.35 times that of N. This light reaction is photoreversible. The quantum yield ratio for the forward and back reaction is 0.18 +/- 0.02. The maximum photo steady state concentration of KN is about 0.24. The N intermediate was also trapped in water suspensions of purple membranes at neutral pH and low salt concentration by illumination at lambda greater than 620 nm during cooling. In addition to N another intermediate absorbing in the red (maximum at 610-620 nm) was accumulated in smaller amounts. It is not photoactive at -196 degrees C and apparently is the O intermediate or a photoproduct of N.     

The transverse location of the retinal chromophore in the purple membrane by diffusion-enhanced energy transfer

We have used fluorescence energy transfer in the rapid-diffusion limit (RDL) to estimate the trans-membrane depth of retinal in the purple membrane (PM). Chelates of Tb(III) are excellent energy donors for the retinal chromophore of PM, having a maximum Ro value for F?rster energy transfer of approximately 62 A (assuming a donor quantum yield of 1). Energy transfer rates were measured from the time-resolved emission kinetics of the donor. The distance of closest approach between chelates and the chromophore was estimated by simulating RDL energy-transfer rate constants according to geometric models of either PM sheets or membrane vesicles. The apparent rate constant for RDL energy transfer between Tb(III)HED3A and retinal in PM sheets is 1.5(+/- 0.1) x 10(6) M-1 s-1, corresponding to a depth of approximately 10 +/- 2 A for the retinal chromophore. Cell envelope vesicles (CEVs) from Halobacterium halobium were studied by using RDL energy transfer to assess the proximity of retinal to either the extracellular or intracellular face of the PM. The estimated depth of retinal from the extravesicular face of the PM is 10 +/- 3 A, based on the RDL energy-transfer rate constant. Energy-transfer levels to retinal in the PM were estimated by an indirect method with energy donors trapped in the inner-aqueous space of CEVs. The rate constants derived for this arrangement are too low to be consistent with the shortest depth of retinal deduced for PM sheets. Thus, the intravesticular face of CEVs, corresponding to the cytoplasmic face of cells, is the more distant surface from the chromophore of bacteriorhodopsin.     

The fluorescence from the chromophore of the purple membrane protein.

The fluorescence from the purple membrane protein (PM) of Halobacterium halobium and its relation to the primary photochemical events have been studied. The emission spectrum at 77 degrees K has structure, with peaks at 680, 710-715, and 730-735 nm. The excitation spectrum shows a single peak centered at 580 nm. This and a comparison of the fluorescence intensity at 77 degrees K under a variety of conditions with the amounts of the bathoproduct (or K, the only photoproduct seen at this temperature) formed suggest that the source of the fluorescence is the purple membrane itself, not the photoproduct. From the difference in several of their properties, we suggest that the fluorescing state of the pigment is different from the excited state which leads to photoconversion.     

The quantum efficiency of proton pumping by the purple membrane of Halobacterium halobium.

The quantum yield of H+ release in purple membrane (PM) sheets, and H+ uptake in phospholipid (egg phosphatidylcholine, PC) vesicles containing PM, was measured in single turnover light flashes using a pH-sensitive dye, p-nitrophenol, with rhodopsin as an actinometer. We have also calculated the ratio of H+ released per M412 formed (an unprotonated Shiff-base intermediate formed during the photocycle). In PM sheets, the quantum yield of H+ release depends on the medium. The quantum yield of M412 is independent of salt concentration. The ratio H+/M412 is approximately 1.8 M KC; and approximately 0.64 in 10 mM KCl. Direct measurements of the quantum yield of H+ give approximately 0.7 when the PM is suspended in 0.5 M KC; and 0.25 in 10 mM KCl. Using a quantum yield for M412 formation of 0.3 (Becher and Ebrey, 1977 Biophys J. 17:185.), these measurements also give a H+/M412 approximately 2 at high salt. In PM/PC vesicles, the H+/M412 is approximately 2 at all salt concentrations. The M412 decay is biphasic and the dye absorption change is monophasic. The dissipation of the proton gradient is very slow, taking on the order of seconds. Addition of nigericin (H+/K+ antiporter) drastically reduces the pH changes observed in PM/PC vesicles. This and the observation that the proton relaxation time is much longer than the photochemical cycling time suggest that the protons are pumped across the membrane and there is no contribution as a result of reversible binding and release of protons on just one side of the membrane.     

Picosecond and steady state, variable intensity and variable temperature emission spectroscopy of bacteriorhodopsin.

The bacteriorhodopsin emission lifetime at 77 degrees K has been obtained for different regions of the emission spectrum with single-pulse excitation. The data under all conditions yield a lifetime of 60 +/- 15 ps. Intensity effects on this lifetime have been ruled out by studying the relative emission amplitude as a function of the excitation pulse energy. We relate our lifetime to previously reported values at other temperatures by studying the relative emission quantum efficiency as a function of temperature. These variable temperature studies have indicated that an excited state with an emission maximum at 670 nm begins to contribute to the spectrum as the temperature is lowered. Within our experimental error the picosecond data seem to suggest that this new emission may arise from a minimum of the same electronic state responsible for the 77 degrees K emission at 720 nm. A correlation is noted between a 1.0-ps formation time observed in absorption by Ippen et al. (Ippen, E.P., C.V. Shank, A. Lewis, and M.A. Marcus. 1978. Subpicosecond spectroscopy of bacteriorhodopsin. Science [wash. D.C.]. 200:1279-1281 and a time extrapolated from relative quantum efficiency measurements and the 77 degrees K fluorescence lifetime that we report.     

Excitation spectra for photosystem I and photosystem II in chloroplasts and the spectral characteristics of the distributions of quanta between the two photosystems.

The parameters listed in the title were determined within the context of a model for the photochemical apparatus of photosynthesis. The fluorescence of variable yield at 750 nm at -196 degrees C is due to energy transfer from Photosystem II to Photosystem I. Fluorescence excitation spectra were measured at -196 degrees C at the minimum, FO, level and the maximum, FM, level of the emission at 750 nm. The difference spectrum, FM-FO, which represents the excitation spectrum for FV is presented as a pure Photosystem II excitation spectrum. This spectrum shows a maximum at 677 nm, attributable to the antenna chlorophyll a of Photosystem II units, with a shoulder at 670 nm and a smaller maximum at 650 nm, presumably due to chlorophyll a and chlorophyll b of the light-harvesting chlorophyll complex. Fluoresence at the FO level at 750 nm can be considered in two parts; one part due to the fraction of absorbed quanta, alpha, which excites Photosystem I more-or-less directly and another part due to energy transfer from Photosystem II to Photosystem I. The latter contribution can be estimated from the ratio of FO/FV measured at 692 nm and the extent of FV at 750 nm. According to this procedure the excitation spectrum of Photosystem I at -196 degrees C was determined by subtracting 1/3 of the excitation spectrum of FV at 750 nm from the excitation spectrum of FO at 750 nm. The spectrum shows a relatively sharp maximum at 681 nm due to the antenna chlorophyll a of Photosystem I units with probably some energy transfer from the light-harvesting chlorophyll complex. The wavelength dependence of alpha was determined from fluorescence measurements at 692 and 750 nm at -196 degrees C. Alpha is constant to within a few percent from 400 to 680 nm, the maximum deviation being at 515 nm where alpha shows a broad maximum increasing from 0.30 to 0.34. At wavelengths between 680 and 700 nm, alpha increases to unity as Photosystem I becomes the dominant absorber in the photochemical apparatus.     

Aspartate 75 mutation in sensory rhodopsin II from Natronobacterium pharaonis does not influence the production of the K-like intermediate, but strongly affects its relaxation pathway

The early steps in the photocycle of the aspartate 75-mutated sensory rhodopsin II from Natrobacterium pharaonis (pSRII-D75N) were studied by time-resolved laser-induced optoacoustic spectroscopy combined with quantum yield determinations by flash photolysis with optical detection. Similar to the case of pSRII-WT, excitation of pSRII-D75N produces in subnanosecond time a K-like intermediate. Different to the case of K in pSRII-WT, in pSRII-D75N there are two K states. K(E) decays into K(L) with a lifetime of 400 ns (independent of temperature in the range 6.5-52 degrees C) which is optically silent under the experimental conditions of our transient absorption experiments. This decay is concomitant with an expansion of 6.5 ml/mol of produced intermediate. This indicates a protein relaxation not affecting the chromophore absorption. For pSRII-D75N reconstituted into polar lipids from purple membrane, the mutation of Asp-75 by the neutral residue Asn affects neither the K(E) production yield (PhiK(e) 0.51 +/- 0.05) nor the energy stored by this intermediate (E(E)K(E) = 91 +/- 11 kJ/mol), nor the expansion upon its production (DeltaV(R,1) = 10 +/- 0.3 ml/mol). All these values are very similar to those previously determined for K with pSRII-WT in the same medium. The millisecond transient species is attributed to K(L) with a lifetime corresponding to that determined by electronic absorption spectroscopy for K(565). The determined energy content of the intermediates as well as the structural volume changes for the various steps afford the calculation of the free energy profile of the phototransformation during the pSRII-D75N photocycle. These data offer insights regarding the photocycle in pSRII-WT. Detergent solubilization of pSRII-D75N affects the sample properties to a larger extent than in the case of pSRII-WT.     

Fluorescent probe 4-dimethylaminochalcone: mechanism of fluorescence quenching in nonpolar media

The reasons for the high sensitivity of the fluorescent probe 4-dimethylaminochalcone (DMC) to nonpolar environment were explored. It was shown that, at room temperature, the fluorescence quantum yield in nonpolar media at 20 degrees C is lower than 0.01 (0.001 in methylcyclohexane). However, as temperature was lowered to -196 degrees C, the yield in methylcyclohexane increased more than 200 times. At the same time, the oscillator strength of absorption transition increased, and the absorption spectrum was shifted to red. These results, together with quantum chemistry calculations suggest that, for fluorescence quenching to occur, some barrier in the DMC molecule, probably the barrier of rotation about C-C bonds, should be overcome. In other words, the quenching is associated with the transition of DMC molecules from a flat conformation (energy minimum) to other, nonflat conformations through rotations about C-C bonds. The phosphorescence of DMC at low temperatures was detected. This suggests that fluorescence quenching is caused by radiationless transitions from the excited singlet level to the ground and triplet levels, and rotation about bonds facilitates these transitions.     

Dependence of the quantum efficiency for photolysis of carboxyhemoglobin on the degree of ligation.

A combined stopped flow-laser photolysis apparatus was used to measure the quantum efficiency for removal of carbon monoxide bound to human hemoglobin as a function of fractional CO saturation. This flow-flash technique allows the properties of partially liganded hemoglobin molecules, which are sparsely populated under equilibrium conditions, to be conveniently studied. Experiments performed at pH 7 and 20 degrees C both in the presence and absence of phosphates gave a similar dependence of quantum efficiency on fractional saturation. The observed quantum efficiency was 0.90 +/- 0.06 at 10% saturation and decreased to 0.47 +/- 0.02 as full saturation was approached. An allosteric model in which Hb(CO)1 has a quantum efficiency of 0.99 while other liganded species have quantum efficiencies of 0.47 was used to produce a good simulation of the results.     

Quantum efficiencies of bacteriorhodopsin photochemical reactions.

Determination of quantum efficiencies of bacteriorhodopsin (bR) photoreactions is an essential step toward a full understanding of its light-driven proton-pumping mechanism. The bR molecules can be photoconverted into and from a K state, which is stable at 110 K. I measured the absorption spectra of pure bR, and the photoequilibrium states of bR and K generated with 420, 460, 500, 510, 520, 540, 560, 570, 580, 590, and 600 nm illumination at 110 K. The fraction of the K population in the photoequilibrium state, fk, is determined by AbR and AK the absorbances of the bR and K states at the excitation wavelengths, and also by phi 1 and phi 2, the quantum efficiencies for the bR to K and K to bR photoconversion: fK = phi 1 AbR/(phi 1AbR + phi 2Ak). By assuming that the ratio phi 1/phi 2 is the same at two different but close wavelengths, for example 570 and 580 nm, the value of phi 1/phi 2 at 570 and 580 nm was determined to be 0.55 +/- 0.02, and the spectrum of the K state was obtained with the peak absorbance at 607 nm. The values of phi 1/phi 2 at the other excitation wavelengths were then evaluated using the known K spectrum, and show almost no dependence on the excitation wavelength within the main band. The result phi 1/phi 2 = 0.55 +/- 0.02 disagrees with those of many other groups. The advantages of this method over others are its minimal assumptions and its straightforward procedure.     

A new methodical approach to the determination of the quantum yield of the photoelectric conversion of electronic excitations in reaction centers of purple bacteria

A new methodical approach has been developed, which enables one to determine with a high precision (approximately 1.5%) the quantum yield of energy conversion in reaction centers isolated from purple bacterium. This parameter for reaction centers from Rhodospirillum rubrum was estimated to be 93.5 +/- 1,5%. Our methodical approach makes it possible to calculate quantum yield values for complete photosystems of purple bacteria.     

A low temperature investigation of the intermediates of the photocycle of light-adapted bacteriorhodopsin. Optical absorption and fluorescence measurements.

Optical absorption and emission measurements have been made on samples of light-adapted purple membrane of Halobacterium halobium at temperatures ranging from 77 K to room temperature. As a result of these experiments a set of equations is given which described thermal and photochemical reactions interrelating various intermediates of the reaction cycle of the chromophore of light-adapted bacteriorhodopsin (BR). Further some specific problems connected to these intermediates have been investigated. Thus the room temperature emission spectrum of bacteriorhodopsin has been found to exhibit a Stokes shift of 3430 cm-1 only, if low excitation intensities are used. The recently detected intermiediate P-BR can be shown to convert thermally into bacteriorhodopsin following a first-order decay with the activation energy delta E = 2.4 +/- 0.2 kcal/mol. The thermal decay of K-BR consists of two exponentials if measured on purple membrane suspensions in a mixture of H2O and glycerol (1 : 1, v/v). A simple procedure is given for trapping the intermediate L-BR at 170 K in a very pure form. M-BR is shown to consist of two species, MI-BR and MII-BR. They are characterized by similar optical absorption spectra but different thermal stability. Further the oscillator strengths corresponding to the long wavelength absorption bands of the intermediates bacteriorhodopsin, K-, L, MI- and MII-BR have been calculated. They have been discussed with respect to the question which of the corresponding absorption spectra show the characteristics of isomerism of the chromophore or simply solvatochromism.     

Fluorescence and phosphorescence of adrenolutin

Adrenolutin 3,5,6-trihydroxy-1-methylindole is an intermediate in the metabolism of adrenaline and in the formation of adrenochromo-melanins. Excitation and emission spectra, quantum yield of the adrenolutin fluorescence in water, D2O, ethanol, methanol, acetone and aqueous phosphate buffer at different pH at 293K temperature are reported. Dependence of the quantum yield of adrenolutin on its concentration are measured. Lifetimes of 0.1 mM adrenolutin in water and ethanol are 32.0 +/- 0.2 ns and 9.2 +/- 0.2 ns respectively. Also fluorescence and phosphorescence spectra of adrenolutin in methanol at 110K are obtained. Degrees of polarization and angles between the dipoles for the three main bands absorption of adrenolutin from measurements at 103K are calculated. Adrenolutin may be classified as one of the most strongly fluorescing metabolites. Broad excitation spectrum and high quantum yields make this compound a potential effective acceptor of excitation energy.     

Energy migration in phycobilisomes

Fluorescence emission and polarization spectra of the phycobilisomes (PBS) of the blue-green alga Nostoc muscorum were measured at 20, -73 and -196 degrees C while exciting at the absorption maximum of each pigment in the PBS. The emission spectra were deconvoluted into a number of Gaussian components and energy migration coefficients and quantum yields of fluorescence for the 8 forms of the phycobilins constituting the PBS were calculated. The overlap integrals and the critical and real distances for the energy transfer in the donor-acceptor pairs were evaluated. The general scheme of the energy transfer in the PBS is proposed according to which there is a homogeneous energy migration within each pigment form and a following effective heterogeneous migration directed from the short wavelength forms via the intermediate ones to the terminal long wavelength acceptors. The transfer passes one or more steps of the energy "staircase" which is formed by the excited levels of the forms. The backward "uphill" energy transfer does not take place. These data and the estimates of the real distances of the energy transfer allowed us to make a conclusion on the regular arrangement of the pigments in the PBS, to determine the distances between the chromophores and their localization in a pigment molecule and the distances between the chromophores of different pigments and thus to specify the structure of the PBS.     

Vibrationally enhanced hydrogen tunneling in the Escherichia coli thymidylate synthase catalyzed reaction

The enzyme thymidylate synthase (TS) catalyzes a complex reaction that involves forming and breaking at least six covalent bonds. The physical nature of the hydride transfer step in this complex reaction cascade has been studied by means of isotope effects and their temperature dependence. Competitive kinetic isotope effects (KIEs) on the second-order rate constant (V/K) were measured over a temperature range of 5-45 degrees C. The observed H/T ((T)V/K(H)) and D/T ((T)V/K(D)) KIEs were used to calculate the intrinsic KIEs throughout the temperature range. The Swain-Schaad relationships between the H/T and D/T V/K KIEs revealed that the hydride transfer step is the rate-determining step at the physiological temperature of Escherichia coli (20-30 degrees C) but is only partly rate-determining at elevated and reduced temperatures. H/D KIE on the first-order rate constant k(cat) ((D)k = 3.72) has been previously reported [Spencer et al. (1997) Biochemistry 36, 4212-4222]. Additionally, the Swain-Schaad relationships between that (D)k and the V/K KIEs reported here suggested that at 20 degrees C the hydride transfer step is the rate-determining step for both rate constants. Intrinsic KIEs were calculated here and were found to be virtually temperature independent (DeltaE(a) = 0 within experimental error). The isotope effects on the preexponential Arrhenius factors for the intrinsic KIEs were A(H)/A(T) = 6.8 +/- 2.8 and A(D)/A(T) = 1.9 +/- 0.25. Both effects are significantly above the semiclassical (no-tunneling) predicted values and indicate a contribution of quantum mechanical tunneling to this hydride transfer reaction. Tunneling correction to transition state theory would predict that these isotope effects on activation parameters result from no energy of activation for all isotopes. Yet, initial velocity measurements over the same temperature range indicate cofactor inhibition and result in significant activation energy on k(cat) (4.0 +/- 0.1 kcal/mol). Taken together, the temperature-independent KIEs, the large isotope effects on the preexponential Arrhenius factors, and a significant energy of activation all suggest vibrationally enhanced hydride tunneling in the TS-catalyzed reaction.     

A new infrared electronic transition of the oxidized primary electron donor in bacterial reaction centers: a way to assess resonance interactions between the bacteriochlorophylls.

The primary electron donor in the reaction center of purple photosynthetic bacteria consists of a pair of bacteriochlorophylls (PL and PM). The oxidized dimer (P+) is expected to have an absorption band in the mid-IR, whose energy and dipole strength depend in part on the resonance interactions between the two bacteriochlorophylls. A broad absorption band with the predicted properties was found in a previously unexplored region of the spectrum, centered near 2600 cm-1 in reaction centers of Rhodobacter sphaeroides and several other species of bacteria that contain bacteriochlorophyll a, and near 2750 cm-1 in Rhodopseudomonas viridis. The band is not seen in the absorption spectrum of the monomeric bacteriochlorophyll cation in solution, and it is missing or much diminished in the reaction centers of bacterial mutants that have a bacteriopheophytin in place of either PL or PM. With the aid of a relatively simple quantum mechanical model, the measured transition energy and dipole strength of the band can be used to solve for the resonance interaction matrix element that causes an electron to move back and forth between PL and PM, and also for the energy difference between states in which a positive charge is localized on either PL or PM. (The absorption band can be viewed as representing a transition between supermolecular eigenstates that are obtained by mixing these basis states.) The values of the matrix element obtained in this way agree reasonably well with values calculated by using semiempirical atomic resonance integrals and the reaction center crystal structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational changes in bacteriorhodopsin studied by infrared attenuated total reflection.

We report on a new method based on Fourier transform infrared (FTIR)-difference spectroscopy for studying the conformational changes occurring during the photocycle of bacteriorhodopsin. Previous studies have been made by measuring the absorbance of an infrared (IR) beam transmitted through a thin hydrated purple membrane film. In contrast, the present study utilizes the technique of attenuated total reflection (ATR). Purple membrane is fixed on the surface of a germanium internal reflection crystal and immersed in a buffer whose pH and ionic composition can be varied. Measurements of the amide I and II absorbance with light polarized parallel and at 45 degrees to the crystal surface reveals that the membrane is highly oriented. An ATR-FTIR-difference spectrum of the light to dark (bR570 to bR548) transition is similar but not identical to the transmittance FTIR-difference spectrum. This disagreement between the two methods is shown to be due in the ATR case to the absorption of transition moments oriented predominantly out of the membrane plane. Raising the pH of La3+ substituted purple membrane films from 6.8 to 8.0 slows the M-decay rate sufficiently so that a bR570 to M412 difference spectrum can be obtained with steady state illumination at room temperature. A comparison of this difference spectrum with that obtained at -23 degrees C using the transmittance method reveals several changes that cannot be attributed to out-of-plane transition moments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy transduction during catalysis by Escherichia coli DNA photolyase.

Native DNA photolyase from Escherichia coli contains 1,5-dihydroFAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate. Quantum yield and action spectral data for thymine dimer repair were obtained by using a novel multiple turnover approach under aerobic conditions. This method assumes that catalysis proceeds via a (rapid-equilibrium) ordered mechanism with light as the second substrate, as verified in steady state kinetic studies. The action spectrum observed with native enzyme matched its absorption spectrum and an action spectrum simulated based on an energy transfer mechanism where dimer repair is initiated either by direct excitation of FADH2 or by pterin excitation followed by singlet-singlet energy transfer to FADH2. The quantum yield observed for dimer repair with native enzyme (phi Native = 0.722 +/- 0.0414) is similar to that observed with enzyme containing only FADH2 (phi EFADH2 = 0.655 +/- 0.0256), as expected owing to the high efficiency of energy transfer from the natural pterin to FADH2 [EET = 0.92]. The quantum yield observed for dimer repair decreased (2.1-fold) when the natural pterin was partially (68.8%) replaced with 5,10-CH(+)-H4folate (phi obs = 0.342 +/- 0.0149). This is consistent with the energy transfer mechanism (phi calc = 0.411 +/- 0.0118) since a 2-fold lower energy transfer efficiency is observed when the natural pterin is replaced with 5,10-CH(+)-H4folate (EET = 0.46) (Lipman & Jorns, 1992). The action spectrum observed for 5,10-CH(+)-H4folate-supplemented enzyme matched a simulated action spectrum which exhibited a small (5 nm) hypsochromic shift as compared with the absorption spectrum (lambda max = 385 nm).(ABSTRACT TRUNCATED AT 250 WORDS)