Immobilization of yeast pyridoxaminephosphate oxidase to halogenoacetyl polysaccharides

Pyridoxaminephosphate oxidase (EC 1.4.3.5, deaminating) that was partially purified about 40-fold from dry baker's yeast was immobilized to iodo- and bromoacetyl polysaccharides. The most effective carrier was an iodoacetyl cellulose, to which almost complete activity of pyridoxine 5'-phosphate oxidase was immobilized in 0.02M potassium phosphate buffer (pH 8.5) containing 2M ammonium sulfate at 4 degrees C. The immobilized enzyme was more stable than the purified, soluble enzyme against heat and pH change. It was confirmed that N-(5'-phosphopyridoxyl)-L-serine was degradedly oxidized to pyridoxal 5'-phosphate and L-serine by the immobilized enzyme as comparable rate as pyridoxine 5'-phosphate, whereas N-(5'-phosphopyridoxyl)-D-serine did not serve as substrate, as in the purified, soluble enzyme.     

Reaction mechanism of alanine racemase from Bacillus stearothermophilus: x-ray crystallographic studies of the enzyme bound with N-(5'-phosphopyridoxyl)alanine

The crystal structures of alanine racemase bound with reaction intermediate analogs, N-(5'-phosphopyridoxyl)-L-alanine (PLP-L-Ala) and N-(5'-phosphopyridoxyl)-D-alanine (PLP-D-Ala), were determined at 2.0-A resolution with the crystallographic R factor of 17.2 for PLP-L-Ala and 16.9 for PLP-D-Ala complexes. They were quite similar not only to each other but also to the structure of the native pyridoxal 5'-phosphate (PLP)-form enzyme; root mean square deviations at Calpha among the three structures were less than 0.28 A. The side chains of the amino acid residues around the PLP-L-Ala and PLP-D-Ala were virtually superimposable on each other as well as on those around PLP of the native holoenzyme. The alpha-hydrogen of the alanine moiety of PLP-L-Ala was located near the OH of Tyr(265)', whereas that of PLP-D-Ala was near the NZ of Lys(39). These support the previous findings that Tyr(265)' and Lys(39) are the catalytic bases removing alpha-hydrogen from L- and D-alanine, respectively. The prerequisite for this two-base mechanism is that the alpha-proton abstracted from the substrate is transferred (directly or indirectly) between the NZ of Lys(39) and the OH of Tyr(265'); otherwise the enzyme reaction stops after a single turnover. Only the carboxylate oxygen atom of either PLP-Ala enantiomer occurred at a reasonable position that can mediate the proton transfer; neither the amino acid side chains nor the water molecules were located in the vicinity. Therefore, we propose a mechanism of alanine racemase reaction in which the substrate carboxyl group directly participates in the catalysis by mediating the proton transfer between the two catalytic bases, Lys(39) and Tyr(265)'. The results of molecular orbital calculation also support this mechanism.     

Evidence that 31P NMR is a sensitive indicator of small conformational changes in the coenzyme of aspartate aminotransferase

The pH dependence of 31P-NMR spectra of pig cytosolic aspartate aminotransferase, containing either N-(5'-phosphopyridoxyl)-L-aspartate or pyridoxal 5'-deoxymethylenephosphonate in place of the normal coenzyme pyridoxal 5'-phosphate, has been analysed. The chemical shifts of phosphopyridoxylaspartate and of pyridoxal 5'-deoxymethylenephosphonate model Schiff base in free solution show pK values of 6.3 and 7.4, attributable to the second deprotonation step of phosphate and phosphonate, respectively. However, these compounds behave very differently when bound to apoaspartate aminotransferase. 31P-NMR spectra of these enzyme derivatives indicate that the phosph(on)ate group remains dianionic throughout the pH range 4-8.5. A clear correlation between apparent pK values obtained from spectrophotometric titration of the coenzyme chromophore and those obtained by 31P NMR indicates that the same ionisation is being reported by both methods. The data are interpreted, on the basis of available crystallographic structures of chicken mitochondrial aspartate aminotransferase, to indicate that in each case the alteration in 31P chemical shift results from a conformational change in the coenzyme 5' side chain, in which one of the structures involves a near-eclipsed pair of bonds. Such a stressed conformation produces slight alterations in bond angles around the phosphorus atom, which in turn cause the observed change in 31P chemical shift. The evidence is taken to indicate that in this case 31P NMR is a sensitive reporter of stress in enzyme-bound pyridoxal 5'-phosphate and its derivatives.     

Gramicidin S: a peptide model for protein glycation and reversal of glycation using nucleophilic amines.

Nonenzymatic glycation of proteins has been implicated in various diabetic complications and age-related disorders. Proteins undergo glycation at the N-terminus or at the epsilon-amino group of lysine residues. Glycation of proteins proceeds through the stages of Schiff base formation, conversion to ketoamine product and advanced glycation end products. Gramicidin S, which has two ornithine residues, was used as a model system to study the various stages of glycation of proteins using electrospray ionization mass spectrometry. The proximity of two ornithine residues in the peptide favors the glycation reaction. Formation of advanced glycation end products and diglycation on ornithine residues in gramicidin S were observed. The formation of Schiff base adduct is reversible, whereas the Amadori rearrangement to the ketoamine product is irreversible. Nucleophilic amines and hydrazines can deglycate the Schiff base adduct of glucose with peptides and proteins. Hydroxylamine, isonicotinic acid hydrazide and aminoguanidine effectively removed glucose from the Schiff base adduct of gramicidin S. Hydroxylamine is more effective in deglycating the adduct compared with isonicotinic acid hydrazide and aminoguanidine. The observation that the hydrazines are effective in deglycating the Schiff base adduct even in the presence of high concentrations of glucose, may have a possible therapeutic application in preventing complications of diabetes mellitus. Hydrazines may be used to distinguish between the Schiff base and the ketoamine products formed at the initial stages of glycation.     

Induction of ornithine decarboxylase in guinea-pig lymphocytes. Synergistic effect of diacylglycerol and calcium

Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5) and trifluoperazine inhibited ornithine decarboxylase induction in lymphocytes activated with phytohemagglutinin or inophore A23187. W-7, a more potent calmodulin antagonist than W-5, suppressed ornithine decarboxylase induction in a higher extent than did W-5. These results suggest that calmodulin may play an important role in ornithine decarboxylase induction in the activated lymphocytes. However, the extent of ornithine decarboxylase induction was greater in cells pretreated with Clostridium phospholipase C and then incubated with ionophore A23187 than in cells incubated with ionophore A23187 without the pretreatment. Moreover, combined treatment of cells with ionophore A23187 and tumor promotor, phorbol 12-myristate 13-acetate, caused synergistic induction of ornithine decarboxylase activity. These results, taken together, suggest that both activations of Ca2+-activated phospholipid-dependent protein kinase by diacylglycerol and of calmodulin-dependent function resulted from an elevation of cytosolic Ca2+ concentration may operate in the induction of ornithine decarboxylase in the activated lymphocytes.     

Decarboxylases involved in polyamine biosynthesis and their inactivation by nitric oxide

Polyamines are ubiquitous cellular components that are involved in normal and neoplastic growth. Polyamine biosynthesis is very highly regulated in mammalian cells by the activities of two key decarboxylases acting on ornithine and S-adenosylmethionine. Recent studies, which include crystallographic analysis of the recombinant human proteins, have provided a detailed knowledge of their structure and function. Ornithine decarboxylase is a PLP-requiring decarboxylase, whereas S-adenosylmethionine decarboxylase (AdoMetDC) contains a covalently bound pyruvate prosthetic group. Both enzymes have a key cysteine residue, which is involved in protonation of the Schiff base intermediate C(alpha) to form the product. These residues, Cys360 in ornithine decarboxylase (ODC) and Cys82 in AdoMetDC, react readily with nitric oxide (NO), which is therefore a potent inactivator of polyamine synthesis. The inactivation of these enzymes may mediate some of the antiproliferative actions of NO.     

Mechanism of inactivation of ornithine decarboxylase by alpha-methylornithine

Ornithine decarboxylase from Lactobacillus 30a is gradually inactivated by treatment with alpha-methylornithine, but activity is restored by treatment of the inactivated enzyme with pyridoxal phosphate. Inactivation of the enzyme is associated with formation of pyridoxamine phosphate and 5-amino-2-pentanone, alpha-Methylornithine is decarboxylated by the enzyme about 6000 times more slowly than is ornithine under the same conditions. These observations provide an explanation for the previously observed inhibition of ornithine decarboxylase by alpha-methylornithine [M. M. Adbel-Monem, N. E. Newton, and C. E. Weeks (1974), J. Med. Chem. 17, 4447]: alpha-Methylornithine undergoes a decarboxylation-dependent transamination as a result of incorrect protonation of the quinoid intermediate which is formed by decarboxylation of the enzyme-bound pyridoxal phosphate-substrate Schiff base. This protonation produces inactive enzyme. Decarboxylation of ornithine by this enzyme produces a small amount of 4-aminobutanal, presumably also by decarboxylation-dependent transamination.     

The antibody-enzyme analogy. Characterization of antibodies to phosphopyridoxyltyrosine derivatives.

Stable analogs of the crucial Schiff base intermediate of enzymatic and nonenzymatic pyridoxal phosphate catalysis have been used as haptens for induction of specific antibodies. N-(5-phosphopyridoxyl)-3'-amino-L-tyrosine and its conformationally distinct cyclized derivative resemble the Schiff base formed upon mixing tyrosine with pyridoxal phosphate. These compounds were covalently coupled to a protein carrier via the 3'-amino group so as to confer a prescribed orientation, with the coenzyme region farthest removed from the carrier. A third antigen, with the phosphopyridoxyl group alone as the hapten, was prepared by linkage of pyridoxal phosphate directly to free amino groups on the carrier protein. Antibodies elicited for each determinant were purified by means of appropriate affinity columns. Antibody heterogeneity was observed in that different species could be separated from a given serum by sequential elution from the affinity columns with 1 M sodium phosphate buffers of pH 7.6, 5.2, 2.6 and 1.5. In assays of quantitative precipitation, inhibition of precipitation, equilibrium dialysis, and fluorescence quenching, antibodies to the phosphopyridoxyltyrosine haptens showed specificity for the phosphorylated form of the coenzyme and binding activity for both the coenzyme and tyrosine portions of the hapten. Antibodies to the phosphopyridoxyl groups alone did not display a similar reactivity toward the tyrosine portion of the complex haptens. The cyclic and noncyclic conformations of the hapten were serologically distinct, as antibody to each reacted preferentially with the homologous form.     

The chemical synthesis of N-[1-(2-naphthol)]-phosphatidylethanolamine, a fluorescent phospholipid for excited-state proton transfer studies.

A procedure for the preparation of N-[1-(2-naphthol)]-phosphatidylethanolamine (NAPH-PE) has been developed. The synthesis is based on the Schiff base formation between the NH2 of the phospholipid and the aldehyde moiety of 2-hydroxy-1-naphthaldehyde. Then selective reduction of the imine is used to obtain the stable secondary amine, NAPH-PE. Formation of the intermediate Schiff base and the final product is confirmed by 13C- and 1H-NMR. Similar to free 2-naphthol, the excited-state pKa (pKa*) of its phospholipid derivative appears to be significantly lower than the ground-state pKa. At pH 7.4, the excitation spectrum of NAPH-PE shows no deprotonated species in the ground-state, while the emission spectrum presents a significant contribution of this species. Thus the fluorescent phospholipid exhibits the typical behavior of excited-state proton-transfer probes. NAPH-PE is found to incorporate in dimyristoyllecithin (DML) vesicles. The emission spectrum of the probe inserted in the liposomes is affected by acetate used as a proton acceptor. These properties should also be manifest in other lipid bilayers (e.g., plasma membranes of cells) and used for excited-state proton transfer studies.     

Synthesis and characterization of technetium(V) complexes with tridentate schiff base ligands. X-ray crystal structure of chloro[N-(2-hydroxyphenyl)salicylideneiminato]oxotechnetium(V)

Five-coordinate technetium(V) complexes of the form TcO(L)Cl where L is one of the two tridentate Schiff base ligands N-(2-oxidophenyl)salicylideneiminate or N-(2-mercaptophenyl)salicylideneiminate have been synthesized and characterized. These neutral complexes precipitate from methanol upon reaction of the Schiff base ligand with TcOCl₄^?. A single crystal X-ray structure determination shows that the chloro [[N-(2-oxidophenyl)salicylideneiminato](2?)-N,O,O′]oxotechnetium(V) complex, [TcO(C₁₃H₉NO₂)Cl], formula weight 362, has a distorted square pyramidal coordination geometry with the oxo ligand in the axial position. The steric requirements of the oxo group cause the Tc atom to be displayed 0.67 Å out of the mean equatorial plane of the other four donor atoms. This complex crystallizes in the monoclinic space group P2₁/a with a = 13.423(6) Å, b = 12.570(5) Å, c = 7.769(3) Å, β = 106.53(5)°, V = 1256.7(9) ų, and Z = 4. The structure has been refined to R = 0.047 for 1775 observed reflections.     

Modeling of the spatial structure of eukaryotic ornithine decarboxylases.

We used sequence and structural comparisons to determine the fold for eukaryotic ornithine decarboxylase, which we found is related to alanine racemase. These enzymes have no detectable sequence identity with any protein of known structure, including three pyridoxal phosphate-utilizing enzymes. Our studies suggest that the N-terminal domain of ornithine decarboxylase folds into a beta/alpha-barrel. Through the analysis of known barrel structures we developed a topographic model of the pyridoxal phosphate-binding domain of ornithine decarboxylase, which predicts that the Schiff base lysine and a conserved glycine-rich sequence both map to the C-termini of the beta-strands. Other residues in this domain that are likely to have essential roles in catalysis, substrate, and cofactor binding were also identified, suggesting that this model will be a suitable guide to mutagenic analysis of the enzyme mechanism.     

Isotope effect studies of the pyruvate-dependent histidine decarboxylase from Lactobacillus 30a

The decarboxylation of histidine by the pyruvate-dependent histidine decarboxylase of Lactobacillus 30a shows a carbon isotope effect of k12/k13 = 1.0334 +/- 0.0005 and a nitrogen isotope effect k14/k15 = 0.9799 +/- 0.0006 at pH 4.8, 37 degrees C. The carbon isotope effect is slightly increased by deuteriation of the substrate and slightly decreased in D2O. The observed nitrogen isotope effect indicates that the imine nitrogen in the substrate-Schiff base intermediate complex is ordinarily protonated, and the pH dependence of the carbon isotope effect indicates that both protonated and unprotonated forms of this intermediate are capable of undergoing decarboxylation. As with the pyridoxal 5'-phosphate dependent enzyme, Schiff base formation and decarboxylation are jointly rate-limiting, with the intermediate histidine-pyruvate Schiff base showing a decarboxylation/Schiff base hydrolysis ratio of 0.5-1.0 at pH 4.8. The decarboxylation transition state is more reactant-like for the pyruvate-dependent enzyme than for the pyridoxal 5'-phosphate dependent enzyme. These studies find no particular energetic or catalytic advantage to the use of pyridoxal 5'-phosphate over covalently bound pyruvate in catalysis of the decarboxylation of histidine.     

FTIR spectroscopy of the M photointermediate in pharaonis rhoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. During the photocycle of ppR, the Schiff base of the retinal chromophore is deprotonated upon formation of the M intermediate (ppR(M)). The present FTIR spectroscopy of ppR(M) revealed that the Schiff base proton is transferred to Asp-75, which corresponds to Asp-85 in a light-driven proton-pump bacteriorhodopsin (BR). In addition, the C==O stretching vibrations of Asn-105 were assigned for ppR and ppR(M). The common hydrogen-bonding alterations in Asn-105 of ppR and Asp-115 of BR were found in the process from photoisomerization (K intermediate) to the primary proton transfer (M intermediate). These results implicate similar protein structural changes between ppR and BR. However, BR(M) decays to BR(N) accompanying a proton transfer from Asp-96 to the Schiff base and largely changed protein structure. In the D96N mutant protein of BR that lacks a proton donor to the Schiff base, the N-like protein structure was observed with the deprotonated Schiff base (called M(N)) at alkaline pH. In ppR, such an N-like (M(N)-like) structure was not observed at alkaline pH, suggesting that the protein structure of the M state activates its transducer protein.     

Phototransduction by vertebrate ultraviolet visual pigments: protonation of the retinylidene Schiff base following photobleaching

The photochemical and subsequent thermal reactions of the mouse short-wavelength visual pigment (MUV) were studied by using cryogenic UV-visible and FTIR difference spectroscopy. Upon illumination at 75 K, MUV forms a batho intermediate (lambda(max) approximately 380 nm). The batho intermediate thermally decays to the lumi intermediate (lambda(max) approximately 440 nm) via a slightly blue-shifted intermediate not observed in other photobleaching pathways, BL (lambda(max) approximately 375 nm), at temperatures greater than 180 K. The lumi intermediate has a significantly red-shifted absorption maximum at 440 nm, suggesting that the retinylidene Schiff base in this intermediate is protonated. The lumi intermediate decays to an even more red-shifted meta I intermediate (lambda(max) approximately 480 nm) which in turn decays to meta II (lambda(max) approximately 380 nm) at 248 K and above. Differential FTIR analysis of the 1100-1500 cm(-1) region reveals an integral absorptivity that is more than 3 times smaller than observed in rhodopsin and VCOP. These results are consistent with an unprotonated Schiff base chromophore. We conclude that the MUV-visual pigment possesses an unprotonated retinylidene Schiff base in the dark state, and undergoes a protonation event during the photobleaching cascade.     

Biodegradative ornithine decarboxylase of Escherichia coli. Purification, properties, and pyridoxal 5'-phosphate binding site.

The biodegradative ornithine decarboxylase of Escherichia coli has been purified to apparent homogeneity. At its pH optimum (pH 7.0), the enzyme exists as a dimer of 160,000 molecular weight. Aggregation of the dimer was promoted by lower pH values. The enzyme requires pyridoxal 5'-phosphate for activity. The coenzyme appears to be bound in Schiff base linkage as suggested by spectral studies and inhibition by NaBH4. The following sequence was determined for the coenzyme binding site: Val-His-(epsilon-Pxy)Lys-Gln-Gln-Ala-Gly-Gln. The properties of this enzyme are compared with the other biodegradative amino acid decarboxylases that have been isolated from E. coli.     

The pKa of the protonated Schiff bases of gecko cone and octopus visual pigments.

A visual pigment is composed of retinal bound to its apoprotein by a protonated Schiff base linkage. Light isomerizes the chromophore and eventually causes the deprotonation of this Schiff base linkage at the meta II stage of the bleaching cycle. The meta II intermediate of the visual pigment is the active form of the pigment that binds to and activates the G protein transducin, starting the visual cascade. The deprotonation of the Schiff base is mandatory for the formation of meta II intermediate. We studied the proton binding affinity, pKa, of the Schiff base of both octopus rhodopsin and the gecko cone pigment P521 by spectral titration. Several fluorinated retinal analogs have strong electron withdrawing character around the Schiff base region and lower the Schiff base pKa in model compounds. We regenerated octopus and gecko visual pigments with these fluorinated and other retinal analogs. Experiments on these artificial pigments showed that the spectral changes seen upon raising the pH indeed reflected the pKa of the Schiff base and not the denaturation of the pigment or the deprotonation of some other group in the pigment. The Schiff base pKa is 10.4 for octopus rhodopsin and 9.9 for the gecko cone pigment. We also showed that although the removal of Cl- ions causes considerable blue-shift in the gecko cone pigment P521, it affects the Schiff base pKa very little, indicating that the lambda max of visual pigment and its Schiff base pKa are not tightly coupled.     

Hydrogen-bonding alterations of the protonated Schiff base and water molecule in the chloride pump of Natronobacterium pharaonis

Halorhodopsin is a light-driven chloride ion pump. Chloride ion is bound in the Schiff base region of the retinal chromophore, and unidirectional chloride transport is probably enforced by the specific hydrogen-bonding interaction with the protonated Schiff base and internal water molecules. In this article, we study hydrogen-bonding alterations of the Schiff base and water molecules in halorhodopsin of Natronobacterium pharaonis (pHR) by assigning their N-D and O-D stretching vibrations in D(2)O, respectively. Highly accurate low-temperature Fourier transform infrared spectroscopy revealed that hydrogen bonds of the Schiff base and water molecules are weak in the unphotolyzed state, whereas they are strengthened upon retinal photoisomerization. Halide dependence of the stretching vibrations enabled us to conclude that the Schiff base forms a direct hydrogen bond with Cl(-) only in the K intermediate. Hydrogen bond of the Schiff base is further strengthened in the L(1) intermediate, whereas the halide dependence revealed that the acceptor is not Cl(-), but presumably a water molecule. Thus, it is concluded that the hydrogen-bonding interaction between the Schiff base and Cl(-) is not a driving force of the motion of Cl(-). Rather, the removal of its hydrogen bonds with the Schiff base and water(s) makes the environment around Cl(-) less polar in the L(1) intermediate, which presumably drives the motion of Cl(-) from its binding site to the cytoplasmic domain.     

The antibody-enzyme analogy. Comparison of enzymes and antibodies specific for phosphopyridoxyltyrosine.

Reduced Schiff base compounds of pyridoxal-P and tyrosine, which were used to induce specific antibodies described in the preceding article (V. Raso and B. D. Stolar, Biochemistry, 1975), caused active site-directed inhibition of tyrosine transaminase and tyrosine decarboxylase. The antibodies, studied as analogs of enzymes, were able to bind an unsaturated Schiff base catalytic intermediate, as shown by equilibrium dialysis and absorbance difference spectroscopy. Schiff base formation can proceed while the pyridoxal-P and tyrosine are within the antibody combining site, but the rate of this bimolecular condensation within the sites was not greater than the rate in free solution. Antibody did effect a small rate enhancement for the pyridoxal-P-catalyzed transamination of L-tyrosine. These results are discussed in light of current ideas in the mechanisms of enzyme catalysis.     

Mechanistic basis for suicide inactivation of porphobilinogen synthase by 4,7-dioxosebacic acid, an inhibitor that shows dramatic species selectivity.

4,7-Dioxosebacic acid (4,7-DOSA) is an active site-directed irreversible inhibitor of porphobilinogen synthase (PBGS). PBGS catalyzes the first common step in the biosynthesis of the tetrapyrrole cofactors such as heme, vitamin B(12), and chlorophyll. 4,7-DOSA was designed as an analogue of a proposed reaction intermediate in the physiological PBGS-catalyzed condensation of two molecules of 5-aminolevulinic acid. As shown here, 4,7-DOSA exhibits time-dependent and dramatic species-specific inhibition of PBGS enzymes. IC(50) values vary from 1 microM to 2.4 mM for human, Escherichia coli, Bradyrhizobium japonicum, Pseudomonas aeruginosa, and pea enzymes. Those PBGS utilizing a catalytic Zn(2+) are more sensitive to 4,7-DOSA than those that do not. Weak inhibition of a human mutant PBGS establishes that the inactivation by 4,7-DOSA requires formation of a Schiff base to a lysine that normally forms a Schiff base intermediate to one substrate molecule. A 1.9 A resolution crystal structure of E. coli PBGS complexed with 4,7-DOSA (PDB code ) shows one dimer per asymmetric unit and reveals that the inhibitor forms two Schiff base linkages with each monomer, one to the normal Schiff base-forming Lys-246 and the other to a universally conserved "perturbing" Lys-194 (E. coli numbering). This is the first structure to show inhibitor binding at the second of two substrate-binding sites.     

The role of water in the extracellular half channel of bacteriorhodopsin.

The changes in the photocycle of the wild type and several mutant bacteriorhodopsin (D96N, E204Q, and D212N) were studied on dried samples, at relative humidities of 100% and 50%. Samples were prepared from suspensions at pH approximately 5 and at pH approximately 9. Intermediate M with unprotonated Schiff base was observed at the lower humidity, even in the case where the photocycle in suspension did not contain this intermediate (mutant D212N, high pH). The photocycle of the dried sample stopped at intermediate M1 in the extracellular conformation; conformation change, switching the accessibility of the Schiff base to the cytoplasmic side, and proton transport did not occur. The photocycle decayed slowly by dissipating the absorbed energy of the photon, and the protein returned to its initial bacteriorhodopsin state, through several M1-like substates. These substates presumably reflect different paths of the proton back to the Schiff base, as a consequence of the bacteriorhodopsin adopting different conformations by stiffening on dehydration. All intermediates requiring conformational change were hindered in the dried form. The concentration of intermediate L, which appears after isomerization of the retinal from all-trans to 13-cis, during local relaxation of the protein, was unusually low in dried samples. The lack of intermediates N and O demonstrated that the M state did not undergo a change from the extracellular to the cytoplasmic conformation (M1 to M2 transition), as already indicated by Fourier transform infrared spectroscopy, quasielastic incoherent neutron scattering, and electric signal measurements described in the literature.