Crystal structures of human 108V and 108M catechol O-methyltransferase.

Catechol O-methyltransferase (COMT) plays important roles in the metabolism of catecholamine neurotransmitters and catechol estrogens. The development of COMT inhibitors for use in the treatment of Parkinson's disease has been aided by crystallographic structures of the rat enzyme. However, the human and rat proteins have significantly different substrate specificities. Additionally, human COMT contains a common valine-methionine polymorphism at position 108. The methionine protein is less stable than the valine polymorph, resulting in decreased enzyme activity and protein levels in vivo. Here we describe the crystal structures of the 108V and 108M variants of the soluble form of human COMT bound with S-adenosylmethionine (SAM) and a substrate analog, 3,5-dinitrocatechol. The polymorphic residue 108 is located in the alpha5-beta3 loop, buried in a hydrophobic pocket approximately 16 A from the SAM-binding site. The 108V and 108M structures are very similar overall [RMSD of C(alpha) atoms between two structures (C(alpha) RMSD)=0.2 A], and the active-site residues are superposable, in accord with the observation that SAM stabilizes 108M COMT. However, the methionine side chain is packed more tightly within the polymorphic site and, consequently, interacts more closely with residues A22 (alpha2) and R78 (alpha4) than does valine. These interactions of the larger methionine result in a 0.7-A displacement in the backbone structure near residue 108, which propagates along alpha1 and alpha5 toward the SAM-binding site. Although the overall secondary structures of the human and rat proteins are very similar (C(alpha) RMSD=0.4 A), several nonconserved residues are present in the SAM-(I89M, I91M, C95Y) and catechol- (C173V, R201M, E202K) binding sites. The human protein also contains three additional solvent-exposed cysteine residues (C95, C173, C188) that may contribute to intermolecular disulfide bond formation and protein aggregation.

The V108M mutation decreases the structural stability of catechol O-methyltransferase.

The human gene for catechol O-methyltransferase has a common single-nucleotide polymorphism that results in substitution of methionine (M) for valine (V) 108 in the soluble form of the enzyme (s-COMT). 108M s-COMT loses enzymatic activity more rapidly than 108V s-COMT at physiological temperature, and the 108M allele has been associated with increased risk of breast cancer and several neuropsychiatric disorders. We used circular dichroism (CD), dynamic light scattering, and fluorescence spectroscopy to examine how the 108V/M polymorphism affects the stability of the purified, recombinant protein to heat and guanidine hydrochloride (GuHCl). COMT contains two tryptophan residues, W143 and W38Y, which are located in loops that border the S-adenosylmethionine (SAM) and catechol binding sites. We therefore also studied the single-tryptophan mutants W38Y and W143Y in order to dissect the contributions of the individual tryptophans to the fluorescence signals. The 108V and 108M proteins differed in the stability of both the tertiary structure surrounding the active site, as probed by the fluorescence yields and emission spectra, and their global secondary structure as reflected by CD. With either probe, the midpoint of the thermal transition of 108M s-COMT was 5 to 7 degrees C lower than that of 108V s-COMT, and the free energy of unfolding at 25 degrees C was smaller by about 0.4 kcal/mol. 108M s-COMT also was more prone to aggregation or partial unfolding to a form with an increased radius of hydration at 37 degrees C. The co-substrate SAM stabilized the secondary structure of both 108V and 108M s-COMT. W143 dominates the tryptophan fluorescence of the folded protein and accounts for most of the decrease in fluorescence that accompanies unfolding by GuHCl. While replacing either tryptophan by tyrosine was mildly destabilizing, the lower stability of the 108M variant was retained in all cases.

The histamine N-methyltransferase T105I polymorphism affects active site structure and dynamics.

Histamine N-methyltransferase (HNMT) is the primary enzyme responsible for inactivating histamine in the mammalian brain. The human HNMT gene contains a common threonine-isoleucine polymorphism at residue 105, distal from the active site. The 105I variant has decreased activity and lower protein levels than the 105T protein. Crystal structures of both variants have been determined but reveal little regarding how the T105I polymorphism affects activity. We performed molecular dynamics simulations for both 105T and 105I at 37 degrees C to explore the structural and dynamic consequences of the polymorphism. The simulations indicate that replacing Thr with the larger Ile residue leads to greater burial of residue 105 and heightened intramolecular interactions between residue 105 and residues within helix alpha3 and strand beta3. This altered, tighter packing is translated to the active site, resulting in the reorientation of several cosubstrate-binding residues. The simulations also show that the hydrophobic histamine-binding domain in both proteins undergoes a large-scale breathing motion that exposes key catalytic residues and lowers the hydrophobicity of the substrate-binding site.

Effects of ionizable residues on the absorption spectrum and initial electron-transfer kinetics in the photosynthetic reaction center of Rhodobacter sphaeroides.

Effects of ionizable amino acids on spectroscopic properties and electron-transfer kinetics in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides are investigated by site-directed mutations designed to alter the electrostatic environment of the bacteriochlorophyll dimer that serves as the photochemical electron donor (P). Arginine residues at homologous positions in the L and M subunits (L135 and M164) are changed independently: Arg L135 is replaced by Lys, Leu, Glu, and Gln and Arg M164 by Leu and Glu. Asp L155 also is mutated to Asn, Tyr L164 to Phe, and Cys L247 to Lys and Asp. The mutations at L155, L164, and M164 have little effect on the absorption spectrum, whereas those at L135 and L247 shift the long-wavelength absorption band of P to higher energies. Fits to the ground-state absorption and hole-burned spectra indicate that the blue shift and increased width of the absorption band in the L135 mutants are due partly to changes in the distribution of energies for the zero-phonon absorption line and partly to stronger electron-phonon coupling. The initial electron-transfer kinetics are not changed significantly in most of the mutants, but the time constant increases from 3.0 +/- 0.2 in wild-type RCs to 4.7 +/- 0.2 in C(L247)D and 7.0 +/- 0.3 ps in C(L247)K. The effects of the mutations on the solvation free energies of the product of the initial electron-transfer reaction (P(+)) and the charge-transfer states that contribute to the absorption spectrum ( and ) were calculated by using a distance-dependent electrostatic screening factor. The results are qualitatively in accord with the view that electrostatic interactions of the bacteriochlorophylls with ionized residues of the protein are strongly screened and make only minor contributions to the energetics and dynamics of charge separation. However, the slowing of electron transfer in the Cys L247 mutants and the blue shift of the spectrum in some of the Arg L135 and Cys L247 mutants cannot be explained consistently by electrostatic interactions of the mutated residues with P and B(L); we ascribe these effects tentatively to structural changes caused by the mutations.

Electrostatic interactions in an integral membrane protein.

The effects of charge-charge interactions on the midpoint reduction potential (E(m)()) of the primary electron donor (P) in the photosynthetic reaction center of Rhodobacter sphaeroides were investigated by introducing mutations of ionizable amino acids at selected sites. The mutations were designed to alter the electrostatic environment of P, a bacteriochlorophyll dimer, without greatly affecting its structure or molecular orbitals. Two arginine residues at homologous positions in the L and M subunits [residues (L135) and (M164)], Asp (L155), Tyr (L164), and Cys (L247) were changed independently. Arginine (L135) was replaced by Lys, Leu, Gln, or Glu; Arg (M164), by Leu or Glu; Asp (L155), by Asn; Tyr (L164), by Phe; and Cys (L247), by Lys or Asp. The R(L135)E/C(L247)K double mutant also was made. The shift in the E(m)() of P/P(+) was measured in each mutant and was compared with the effect predicted by electrostatics calculations using several different computational approaches. A simple distance-dependent dielectric screening factor reproduced the effects remarkably well. By contrast, microscopic methods that considered the reaction field in the protein and solvent but did not include explicit counterions overestimated the changes in the E(m)() considerably. Including counterions for the charged residues reduced the calculated effects of the mutations in molecular dynamics calculations. The results show that electrostatic interactions of P with ionizable amino acid residues are strongly screened, and suggest that counterions make major contributions to this screening. The screening also could reflect penetration of water or other relaxations not taken into account because of incomplete sampling of configurational space.

Catecholamines in patients with 22q11.2 deletion syndrome and the low-activity COMT polymorphism.

OBJECTIVE: To investigate catecholamine phenotypes and the effects of a tyrosine hydroxylase inhibitor in individuals with the 22q11.2 deletion syndrome and low-activity catechol-O-methyltransferase (COMT). BACKGROUND: Many persons with the 22q11.2 deletion syndrome suffer severe disability from a characteristic ultrarapid-cycling bipolar disorder and associated "affective storms." One etiologic hypothesis for this condition is that deletion of the COMT gene from one chromosome 22 results in increased catecholamine neurotransmission, particularly if the undeleted chromosome 22 encodes a variant of COMT with low activity. METHODS: In a preliminary study, plasma, urine, and CSF catecholamines and catecholamine metabolites were measured in four teenage patients with a neuropsychiatric condition associated with 22q11.2 deletion and the low-activity COMT polymorphism on the nondeleted chromosome. In these four patients, and an additional institutionalized adult with the condition, an uncontrolled, open-label trial of metyrosine was administered in an attempt to lower catecholamine production and to alleviate symptoms. RESULTS: Mild elevations of baseline CSF homovanillic acid (HVA) were found in three of four patients and a moderate reduction in CSF HVA after metyrosine treatment in the patient with the highest pretreatment concentration. The course of the five patients during the clinical trial is described. CONCLUSIONS: In patients with the 22q11.2 deletion syndrome and low-activity COMT, controlled studies of pharmacologic agents that decrease catecholamine production, block presynaptic catecholamine storage, or enhance S-adenosylmethionine, the cosubstrate of COMT, are warranted.

Reorganization energy of the initial electron-transfer step in photosynthetic bacterial reaction centers.

The reorganization energy (lambda) for electron transfer from the primary electron donor (P*) to the adjacent bacteriochlorophyll (B) in photosynthetic bacterial reaction centers is explored by molecular-dynamics simulations. Relatively long (40 ps) molecular-dynamics trajectories are used, rather than free energy perturbation techniques. When the surroundings of the reaction center are modeled as a membrane, lambda for P* B --> P+ B- is found to be approximately 1.6 kcal/mol. The results are not sensitive to the treatment of the protein's ionizable groups, but surrounding the reaction center with water gives higher values of lambda (approximately 6.5 kcal/mol). In light of the evidence that P+ B- lies slightly below P* in energy, the small lambda obtained with the membrane model is consistent with the speed and temperature independence of photochemical charge separation. The calculated reorganization energy is smaller than would be expected if the molecular dynamics trajectories had sampled the full conformational space of the system. Because the system does not relax completely on the time scale of electron transfer, the lambda obtained here probably is more pertinent than the larger value that would be obtained for a fully equilibrated system.

Excitation energy transfer between the B850 and B875 antenna complexes of Rhodobacter sphaeroides.

Energy transfer between the B850 (LH2) and B875 (LH1) antenna complexes of a mutant strain of Rhodobacter sphaeroides lacking reaction centers is investigated by femtosecond pump-probe spectroscopy at room temperature. Measurements are made at wavelengths between 810 and 910 nm at times extending to 200 ps after selective excitation of either B850 or B875. Assignments of the spectroscopic signals to the two types of antenna complex are made on the basis of measurements in strains that lack either LH1 or LH2 in addition to reaction centers. Energy transfer from excited B850 to B875 proceeds with two time constants, 4.6 +/- 0.3 and 26.3 +/- 1.0 ps, but a significant fraction of the excitations remain in B850 for considerably longer times. The fast step is interpreted as hopping of energy to LH1 from an associated LH2 complex; the slower steps are interpreted as migration of excitations in the LH2 pool preceding transfer to LH1. Transfer of excitations from B875 to B850 could not be detected, possibly suggesting that the average number of LH2 complexes in contact with each LH1 is small.

Ultrafast exciton relaxation in the B850 antenna complex of Rhodobacter sphaeroides.

Spectral changes were measured with femtosecond resolution following low-intensity, broad-band excitation of the peripheral antenna complex of the purple photosynthetic bacterium Rhodobacter sphaeroides. Absorption anisotropy decays also were measured. We identified a 35-fs relaxation of the absorption and emission spectra of the excited state, as well as a 20-fs anisotropy decay. We interpret these results as interlevel relaxation and dephasing, respectively, of extensively delocalized exciton states of the circular bacteriochlorophyll aggregate.

Specific alteration of the oxidation potential of the electron donor in reaction centers from Rhodobacter sphaeroides.

The effects of multiple changes in hydrogen bond interactions between the electron donor, a bacteriochlorophyll dimer, and histidine residues in the reaction center from Rhodobacter sphaeroides have been investigated. Site-directed mutations were designed to add or remove hydrogen bonds between the 2-acetyl groups of the dimer and histidine residues at the symmetry-related sites His-L168 and Phe-M197, and between the 9-keto groups and Leu-L131 and Leu-M160. The addition of a hydrogen bond was correlated with an increase in the dimer midpoint potential. Measurements on double and triple mutants showed that changes in the midpoint potential due to alterations at the individual sites were additive. Midpoint potentials ranging from 410 to 765 mV, compared with 505 mV for wild type, were achieved by various combinations of mutations. The optical absorption spectra of the reaction centers showed relatively minor changes in the position of the donor absorption band, indicating that the addition of hydrogen bonds to histidines primarily destabilized the oxidized state of the donor and had little effect on the excited state relative to the ground state. Despite the change in energy of the charge-separated states by up to 260 meV, the mutant reaction centers were still capable of electron transfer to the primary quinone. The increase in midpoint potential was correlated with an increase in the rate of charge recombination from the primary quinone, and a fit of these data using the Marcus equation indicated that the reorganization energy for this reaction is approximately 400 meV higher than the change in free energy in wild type. The mutants were still capable of photosynthetic growth, although at reduced rates relative to the wild type. These results suggest a role for protein-cofactor interactions--in particular, histidine-donor interactions--in establishing the redox potentials needed for electron transfer in biological systems.

Kinetics and free energy gaps of electron-transfer reactions in Rhodobacter sphaeroides reaction centers.

The rates of the light-driven, electron-transfer reactions in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides are examined in mutant strains in which tyrosine (M)210 is replaced by phenylalanine, isoleucine, or tryptophan. The spectra of the absorbance changes between 700 and 975 nm, following excitation by 0.6-ps pulses at 605 nm, are analyzed globally by singular value decomposition. The spectra measured at room temperature are interpreted in terms of a model in which the excited bacteriochlorophyll dimer (P*) transfers an electron to a bacteriopheophytin (HL) with time constants of 3.5 +/- 0.3, 10.5 +/- 1.0, 16 +/- 2, and 41 +/- 4 ps in wild-type RCs and the Phe, Ile, and Trp mutants, respectively, and an electron then moves from HL- to a quinone (QA) with a time constant of 0.16 ns in wild-type RCs, 0.24 ns in the Phe mutant, and 0.20 ns in the Ile and Trp mutants. The first step speeds up with decreasing temperature in wild-type RCs, remains virtually unchanged in the Phe mutant, and slows down in the Ile and Trp mutants. At 80 K, the signals in the 850-975-nm region include an apparent shift of the stimulated emission or absorption spectrum of P*, with a time constant of 5 ps in the Ile mutant and 13 pcs in the Trp mutant. Most of the electron transfer to HL occurs with time constants of 55 and 155 ps in the Ile and Trp mutants, respectively, and probably occurs from the relaxed form of P*. Electron transfer from the initial state cannot be ruled out, however. Relaxations of P* are not resolved in wild-type RCs or the Phe mutant. The midpoint potential (Em) of the P/P+ redox couple is measured by an electrochemical technique; the Em values are 500 +/- 5, 530 +/- 6, 533 +/- 3, and 552 +/- 10 mV for the wild-type and the Phe, Ile, and Trp mutant RCs, respectively. These values are corroborated by chemical titrations. The free energy change (delta G degrees) associated with formation of the P+HL-radical pair from P* also is determined by measuring the amplitude of fluorescence on the nanosecond time scale after blocking electron transfer from HL- to QA. The free energy of P+HL- is elevated by an amount comparable to that calculated from the increase in the Em of P in the Ile mutant and by about 16 meV more than this in the Phe and Trp mutants.(ABSTRACT TRUNCATED AT 400 WORDS)

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)

Effect of specific mutations of tyrosine-(M)210 on the primary photosynthetic electron-transfer process in Rhodobacter sphaeroides.

We have measured the rate of the initial electron-transfer process as a function of temperature in reaction centers in a native strain of the photosynthetic bacterium Rhodobacter sphaeroides and two mutants generated by site-directed mutagenesis. In the mutants, a tyrosine residue in the vicinity of the primary electron donor and acceptor molecules was replaced by either phenylalanine or isoleucine. The electron-transfer reaction is slower in the mutants and has a qualitatively different dependence on temperature. In native reaction centers the rate increases as the temperature is reduced, in the phenylalanine mutant it is virtually independent of temperature, and in the isoleucine mutant it decreases with decreasing temperature. At 77 K, the electron-transfer reaction is approximately 30 times slower in the isoleucine mutant than in the native. These observations support the view that tyrosine-(M)210 plays an important role in the electron-transfer mechanism. In the isoleucine mutant at low temperatures, the stimulated emission from the excited reaction center undergoes a time-dependent shift to shorter wavelengths.

Electrostatic control of charge separation in bacterial photosynthesis.

Electrostatic interaction energies of the electron carriers with their surroundings in a photosynthetic bacterial reaction center are calculated. The calculations are based on the detailed crystal structure of reaction centers from Rhodopseu-domonas viridis, and use an iterative, self-consistent procedure to evaluate the effects of induced dipoles in the protein and the surrounding membrane. To obtain the free energies of radical-pair states, the calculated electrostatic interaction energies are combined with the experimentally measured midpoint redox potentials of the electron carriers and of bacteriochlorophyll (BChl) and bacteriopheophytin (BPh) in vitro. The P+HL- radical-pair, in which an electron has moved from the primary electron donor (P) to a BPh on the 'L' side of the reaction center (HL), is found to lie approx. 2.0 kcal/mol below the lowest excited singlet state (P*), when the radical-pair is formed in the static crystallographic structure. The reorganization energy for the subsequent relaxation of P+HL- is calculated to be 5.0 kcal/mol, so that the relaxed radical-pair lies about 7 kcal/mol below P*. The unrelaxed P+BL- radical-pair, in which the electron acceptor is the accessory BChl located between P and HL, appears to be essentially isoenergetic with P*.P+BM-, in which an electron moves to the BChl on the 'M' side, is calculated to lie about 5.5 kcal/mol above P*. These results have an estimated error range of +/- 2.5 kcal/mol. They are shown to be relatively insensitive to various details of the model, including the charge distribution in P+, the atomic charges used for the amino acid residues, the boundaries of the structural region that is considered microscopically and the treatments of the histidyl ligands of P and of potentially ionizable amino acids. The calculated free energies are consistent with rapid electron transfer from P* to HL by way of BL, and with a much slower electron transfer to the pigments on the M side. Tyrosine M208 appears to play a particularly important role in lowering the energy of P+BL-. Electrostatic interactions with the protein favor localization of the positive charge of P+ on PM, one of the two BChl molecules that make up the electron donor.

Dispersed polaron simulations of electron transfer in photosynthetic reaction centers.

A microscopic method for simulating quantum mechanical, nuclear tunneling effects in biological electron transfer reactions is presented and applied to several electron transfer steps in photosynthetic bacterial reaction centers. In this "dispersed polaron" method the fluctuations of the protein and the electron carriers are projected as effective normal modes onto an appropriate reaction coordinate and used to evaluate the quantum mechanical rate constant. The simulations, based on the crystallographic structure of the reaction center from Rhodopseudomonas viridis, focus on electron transfer from a bacteriopheophytin to a quinone and the subsequent back-reaction. The rates of both of these reactions are almost independent of temperature or even increase with decreasing temperature. The simulations reproduce this unusual temperature dependence in a qualitative way, without the use of adjustable parameters for the protein's Franck-Condon factors. The observed dependence of the back-reaction on the free energy of the reaction also is reproduced, including the special behavior in the "inverted region."

Microscopic simulation of quantum dynamics and nuclear tunneling in bacterial reaction centers.

The "dispersed polaron" version of the semiclassical trajectory approach is used to evaluate the quantum mechanical nuclear tunneling effects in the charge recombination reaction, P(+)Q(-)→PQ, in photosynthetic bacterial reaction centers, The cclculations are based on the crystallographic structure of reaction centers from Rhodopseudomonas viridis. They succeed in capturing the temperature dependence of the rate constant without using adjustable parameters. This provides the first example of a microscopic simulation of quantum mechanical nuclear tunneling in a biological system.

Design, construction, and use of an electroporator for plant protoplasts and animal cells.

We have designed and constructed an electroporation device capable of efficient transfer of DNA into both plant cell protoplasts and cultured murine lymphocytes. The electroporator design allows various combinations of voltage and capacitance to be used to optimize the electric pulse. Switching of large voltages and currents is accomplished with a silicon-controlled rectifier, yielding excellent reproducibility and long component life. A safety switch is provided to permit complete discharge of the device. Conditions suitable for high levels of transient expression and high frequencies of stable transformation for both plant and animal cell systems have been found.

Radical-pair energetics and decay mechanisms in reaction centers containing anthraquinones, naphthoquinones or benzoquinones in place of ubiquinone.

In reaction centers from Rhodobacter sphaeroides (formerly called Rhodopseudomonas sphaeroides), light causes an electron-transfer reaction that forms the radical pair state (P+I-, or PF) from the initial excited singlet state (P) of a bacteriochlorophyll dimer (P). Subsequent electron transfer to a quinone (Q) produces the state P+Q-. Back electron transfer can regenerate P from P+Q-, giving rise to 'delayed' fluorescence that decays with approximately the same lifetime as P+Q-. The free-energy difference between P+Q- and P can be determined from the initial amplitude of the delayed fluorescence. In the present work, we extracted the native quinone (ubiquinone) from Rps. sphaeroides reaction centers, and replaced it by various anthraquinones, naphthoquinones, and benzoquinones. We found a rough correlation between the halfwave reduction potential (E1/2) of the quinone used for reconstitution (as measured polarographically in dimethylformamide) and the apparent free energy of the state P+Q- relatively to P. As the E1/2 of the quinone becomes more negative, the standard free-energy gap between P+Q- and P decreases. However, the correlation is quantitatively weak. Apparently, the effective midpoint potentials (Em) of the quinones in situ depend subtly on interactions with the protein environment in the reaction center. Using the value of the Em for ubiquinone determined in native reaction centers as a reference, and the standard free energies determined for P+Q- in reaction centers reconstituted with other quinones, the effective Em values of 12 different quinones in situ are estimated. In native reaction centers, or in reaction centers reconstituted with quinones that give a standard free-energy gap of more than about 0.8 eV between P+Q- and P*, charge recombination from P+Q- to the ground state (PQ) occurs almost exclusively by a temperature-insensitive mechanism, presumably electron tunneling. When reaction centers are reconstituted with quinones that give a free-energy gap between P+Q- and P* of less than 0.8 with quinones that give a free-energy gap between P+Q- and P* of less than 0.8 eV, part or all of the decay proceeds through a thermally accessible intermediate. There is a linear relationship between the log of the rate constant for the decay of P+Q- via the intermediate state and the standard free energy of P+Q-. The higher the free energy, the faster the decay. The kinetic and thermodynamic properties of the intermediate appear not to depend strongly on the quinone used for reconstitution, indicating that the intermediate is probably not simply an activated form of P+Q-.(ABSTRACT TRUNCATED AT 400 WORDS)