Kazuo Yoshihara

Photoactive yellow protein (PYP) belongs to the novel group of eubacterial photoreceptor proteins. To fully understand its light signal transduction mechanisms, elucidation of the intramolecular pathway of the internal proton is indispensable because it closely correlates with the changes in the hydrogen-bonding network, which is likely to induce the conformational changes. For this purpose, the vibrational modes of PYP and its photoproduct were studied by Fourier transform infrared spectroscopy at −40 °C. The vibrational modes characteristic for the anionic p-coumaryl chromophore (Kim, M., Mathies, R. A., Hoff, W. D., and Hellingwerf, K. J. (1995)Biochemistry34, 12669–12672) were observed at 1482, 1437, and 1163 cm−1 for PYP. However, the bands corresponding to these modes were not observed for PYPM, the blue-shifted intermediate, but the 1175 cm−1 band characteristic of the neutral p-coumaryl chromophore was observed, indicating that the phenolic oxygen of the chromophore is protonated in PYPM. A 1736 cm−1 band was observed for PYP, but the corresponding band for PYPM was not. Because it disappeared in the Glu-46 → Gln mutant of PYP, this band was assigned to the C=O stretching mode of the COOH group of Glu-46. These results strongly suggest that the proton at Glu-46 is transferred to the chromophore during the photoconversion from PYP to PYPM. Photoactive yellow protein (PYP) belongs to the novel group of eubacterial photoreceptor proteins. To fully understand its light signal transduction mechanisms, elucidation of the intramolecular pathway of the internal proton is indispensable because it closely correlates with the changes in the hydrogen-bonding network, which is likely to induce the conformational changes. For this purpose, the vibrational modes of PYP and its photoproduct were studied by Fourier transform infrared spectroscopy at −40 °C. The vibrational modes characteristic for the anionic p-coumaryl chromophore (Kim, M., Mathies, R. A., Hoff, W. D., and Hellingwerf, K. J. (1995)Biochemistry34, 12669–12672) were observed at 1482, 1437, and 1163 cm−1 for PYP. However, the bands corresponding to these modes were not observed for PYPM, the blue-shifted intermediate, but the 1175 cm−1 band characteristic of the neutral p-coumaryl chromophore was observed, indicating that the phenolic oxygen of the chromophore is protonated in PYPM. A 1736 cm−1 band was observed for PYP, but the corresponding band for PYPM was not. Because it disappeared in the Glu-46 → Gln mutant of PYP, this band was assigned to the C=O stretching mode of the COOH group of Glu-46. These results strongly suggest that the proton at Glu-46 is transferred to the chromophore during the photoconversion from PYP to PYPM. Photoactive yellow protein (PYP) 1The abbreviations used are: PYP, photoactive yellow protein from E. halophila; FTIR, Fourier transform infrared. (λmax = 446 nm) (1Meyer T.E. Biochim. Biophys. Acta. 1985; 806: 175-183Crossref PubMed Scopus (368) Google Scholar) is proposed to be a photoreceptor protein for the negative phototaxis observed in the purple phototrophic bacterium, Ectothiorhodospira halophila (2Sprenger W.W. Hoff W.D. Armitage J.P. Hellingwerf K.J. J. Bacteriol. 1993; 175: 3096-3104Crossref PubMed Scopus (350) Google Scholar). PYP belongs to the novel group of photoreceptor proteins (3Koh M. Van Driessche G. Samyn B. Hoff W.D. Meyer T.E. Cusanovich M.A. Van Beeumen J.J. Biochemistry. 1996; 35: 2526-2534Crossref PubMed Scopus (49) Google Scholar, 4Kort R. Hoff W.D. Van West M. Kroon A.R. Hoffer S.M. Vlieg K.H. Crielaard W. Van Beeumen J.J. Hellingwerf K.J. EMBO J. 1996; 15: 3209-3218Crossref PubMed Scopus (122) Google Scholar) whose structures are quite different from those of the other photoreceptor proteins studied so far. Namely, the protein moiety of PYP has anα/β fold structure (5Borgstahl G.E.O. Williams D.R. Getzoff E.D. Biochemistry. 1995; 34: 6278-6287Crossref PubMed Scopus (440) Google Scholar) composed of 125 amino acids (6Van Beeumen J.J. Devreese B.V. Van Bun S.M. Hoff W.D. Hellingwerf K.J. Meyer T.E. McRee D.E. Cusanovich M.A. Protein Sci. 1993; 2: 1114-1125Crossref PubMed Scopus (83) Google Scholar, 7Baca M. Borgstahl G.E.O. Boissinot M. Burke P.M. Williams D.R. Slater K.A. Getzoff E.D. Biochemistry. 1994; 33: 14369-14377Crossref PubMed Scopus (291) Google Scholar). The chromophore is a p-coumaric acid (7Baca M. Borgstahl G.E.O. Boissinot M. Burke P.M. Williams D.R. Slater K.A. Getzoff E.D. Biochemistry. 1994; 33: 14369-14377Crossref PubMed Scopus (291) Google Scholar, 8Hoff W.D. Düx P. Hård K. Devreese B. Nugteren-Roodzant I.M. Crielaard W. Boelens R. Kaptein R. Van Beeumen J. Hellingwerf K.J. Biochemistry. 1994; 33: 13959-13962Crossref PubMed Scopus (264) Google Scholar, 9Imamoto Y. Ito T. Kataoka M. Tokunaga F. FEBS Lett. 1995; 374: 157-160Crossref PubMed Scopus (124) Google Scholar) bound to a cysteine residue via a thioester bond. PYP absorbs a photon and enters the photocycle. We have analyzed the photocycle of PYP in detail by low temperature spectroscopy and identified several intermediates (10Imamoto Y. Kataoka M. Tokunaga F. Biochemistry. 1996; 35: 14047-14053Crossref PubMed Scopus (127) Google Scholar). Irradiation of PYP at −190 °C yields PYPB (λmax = 489 nm) and PYPH (λmax = 442 nm), which are thermally converted to PYPL (λmax = 456 nm) through PYPBL (λmax = 400 nm) and PYPHL(λmax = 447 nm), respectively. The two pathways beginning with PYPB and PYPH join at PYPL and revert to PYP. Flash photolysis at ambient temperature identified two intermediates, pR (λmax = 465 nm) and pB (λmax = 355 nm) (11Meyer T.E. Yakali E. Cusanovich M.A. Tollin G. Biochemistry. 1987; 26: 418-423Crossref PubMed Scopus (287) Google Scholar, 12Hoff W.D. Van Stokkum I.H.M. Van Ramesdonk H.J. Van Brederode M.E. Brouwer A.M. Fitch J.C. Meyer T.E. Van Grondelle R. Hellingwerf K.J. Biophys. J. 1994; 67: 1691-1705Abstract Full Text PDF PubMed Scopus (252) Google Scholar). pR is formed within 10 ns after flash excitation. It decays to pB over a submillisecond time scale and reverts to PYP within 1 s. It has been demonstrated that pR is the same species as PYPL (10Imamoto Y. Kataoka M. Tokunaga F. Biochemistry. 1996; 35: 14047-14053Crossref PubMed Scopus (127) Google Scholar) (In this paper, pR and pB are called PYPL and PYPM, respectively, to avoid confusion) and that PYPL is accumulated by irradiation of PYP at −80 °C (10Imamoto Y. Kataoka M. Tokunaga F. Biochemistry. 1996; 35: 14047-14053Crossref PubMed Scopus (127) Google Scholar, 13Imamoto Y. Tokunaga F. Fujimori M. Kataoka M. Photomed. Photobiol. 1995; 17: 111-115Google Scholar). However, the precursors of PYPLhave not been discovered by flash photolysis at room temperature. Recent studies have clarified some details of the events that take place during the PYP photocycle. Crystallography at 1.4-Å resolution (7Baca M. Borgstahl G.E.O. Boissinot M. Burke P.M. Williams D.R. Slater K.A. Getzoff E.D. Biochemistry. 1994; 33: 14369-14377Crossref PubMed Scopus (291) Google Scholar) and resonance Raman spectroscopy (14Kim M. Mathies R.A. Hoff W.D. Hellingwerf K.J. Biochemistry. 1995; 34: 12669-12672Crossref PubMed Scopus (139) Google Scholar) have shown that the phenolic oxygen of the chromophore of PYP is deprotonated in the dark state. The photocycle is initiated by photon absorption, which involves isomerization of the ethylenic bond of the chromophore (15Kort R. Vonk H. Xu X. Hoff W.D. Crielaard W. Hellingwerf K.J. FEBS Lett. 1996; 382: 73-78Crossref PubMed Scopus (204) Google Scholar). During the photocycle, observed proton uptake and release correspond with the formation and decay of PYPM (16Meyer T.E. Cusanovich M.A. Tollin G. Arch. Biochem. Biophys. 1993; 306: 515-517Crossref PubMed Scopus (48) Google Scholar). The largely blue-shifted absorption spectrum of PYPMsuggests that the chromophore/protein interaction of PYPMis quite different from that of PYP. Therefore, elucidation of the chromophore structure of PYPM is essential to understand the photocycle of PYP. For the spectral blue-shift of PYPM, the following explanations would be possible: (i) the phenolic oxygen of the chromophore is protonated; (ii) the conjugated double bond system is broken by extreme distortion of the chromophore (25Kakitani H. Kakitani T. Rodman H. Honig B. Photochem. Photobiol. 1985; 41: 471-479Crossref PubMed Scopus (110) Google Scholar); and (iii) the π electrons of the conjugated double bond system are localized by a nearby positive charge (e.g. Arg-52) (26Honig B. Greenberg A.D. Dinur U. Ebrey T.G. Biochemistry. 1976; 15: 4593-4599Crossref PubMed Scopus (208) Google Scholar). The first explanation is the most simple and plausible, but there has been no previous experimental evidence to support it, and the others could not be excluded. In the dark state, the phenolic oxygen of the chromophore interacts with the OH groups of Tyr-42 and Glu-46 by hydrogen bonds (5Borgstahl G.E.O. Williams D.R. Getzoff E.D. Biochemistry. 1995; 34: 6278-6287Crossref PubMed Scopus (440) Google Scholar). As shown in retinal protein systems, the changes in the hydrogen-bonding network centering on the chromophore closely correlates with the protein conformational changes (17Maeda A. Isr. J. Chem. 1995; 35: 387-400Crossref Scopus (133) Google Scholar). Therefore, it is of importance to study the internal proton movement around the chromophore and nearby amino acid residues to understand the light signal transduction mechanism of PYP. Recently it has been reported that the C=O stretching mode of the COOH group disappears on the formation of PYPM (18Xie A. Hoff W.D. Kroon A.R. Hellingwerf K.J. Biochemistry. 1996; 35: 14671-14678Crossref PubMed Scopus (183) Google Scholar). They reasoned that Glu-46 donates a proton to the chromophore, based on the fact that Glu-46 has a unique COOH group embedded in the protein. This idea arose on elucidation of the tertiary structure of PYP (5Borgstahl G.E.O. Williams D.R. Getzoff E.D. Biochemistry. 1995; 34: 6278-6287Crossref PubMed Scopus (440) Google Scholar) and seems reasonable. However, two vital pieces of experimental evidence required to support this conclusion have never been reported: one is that the chromophore of PYPM is protonated, and the other is assignment of the C=O stretching mode. Recent progress in experimental techniques made it possible to prepare PYP with an isotope-labeled chromophore (9Imamoto Y. Ito T. Kataoka M. Tokunaga F. FEBS Lett. 1995; 374: 157-160Crossref PubMed Scopus (124) Google Scholar) and a site-directed mutant of PYP (19Mihara, K., Hisatomi, O., Imamoto, Y., Kataoka, M., and Tokunaga, F. (1997) J. Biochem. (Tokyo), in press.Google Scholar, 27Genick U.K. Devanathan S. Meyer T.E. Canestrelli I.L. Williams E. Cusanovich M.A. Tollin G. Getzoff E.D. Biochemistry. 1997; 36: 8-14Crossref PubMed Scopus (137) Google Scholar). These techniques have enabled assignment of the vibrational modes. In the present study, to study the movement of the proton around the chromophore and nearby amino acid residues, the vibrational modes of the chromophore and COOH group of PYP and its intermediates were analyzed by low temperature FTIR spectroscopy. The first experimental findings indicating proton transfer from Glu-46 to the chromophore during the photocycle are presented. PYP was isolated from E. halophila BN 9626 according to previously reported methods (1Meyer T.E. Biochim. Biophys. Acta. 1985; 806: 175-183Crossref PubMed Scopus (368) Google Scholar, 10Imamoto Y. Kataoka M. Tokunaga F. Biochemistry. 1996; 35: 14047-14053Crossref PubMed Scopus (127) Google Scholar). The Glu-46 → Gln mutant of PYP (E46Q) was expressed by Escherichia coli and reconstituted with p-coumaric anhydride (19Mihara, K., Hisatomi, O., Imamoto, Y., Kataoka, M., and Tokunaga, F. (1997) J. Biochem. (Tokyo), in press.Google Scholar).p-Coumaric-8-13C-acid was prepared byp-hydroxybenzaldehyde and triethylphosphonoacetate-2-13C followed by alkaline hydrolysis of the ester. 13C-Labeled PYP was prepared by reconstitution of PYP with 13C-labeledp-coumaric anhydride and apoPYP (9Imamoto Y. Ito T. Kataoka M. Tokunaga F. FEBS Lett. 1995; 374: 157-160Crossref PubMed Scopus (124) Google Scholar). They were then desalted by dialysis and applied to a small DEAE-Sepharose column (Pharmacia Biotech Inc.). After washing the column with 10 mmphosphate buffer (pH 7.2), PYP was eluted with a linear gradient of NaCl (100–200 mm) in the same buffer. PYP was then concentrated with an ultrafiltration membrane (Centriprep 10, Amicon) and diluted with 10 mm phosphate buffer. Dilution and concentration steps were repeated several times to remove NaCl. Finally, PYP was concentrated to 3–4 mg/ml. A 20 μl sample was placed on a BaF2 window (10 mm in diameter) and dried under a gentle stream of N2 gas. The dried sample was sealed using a silicon rubber spacer and another BaF2 window and set in the sample cell holder. Before sealing, 0.2 μl of H2O or D2O was put inside the spacer for hydration or deuteration of PYP. The sample cell holder was mounted in an optical cryostat (DN1704, Oxford) connected to a temperature controller (ITC502, Oxford). Absorption spectra in the UV-visible region were recorded with a Hitachi U-3210 recording spectrophotometer. Infrared spectra were recorded with a Horiba FT-210 Fourier transform infrared spectrophotometer equipped with an MCT detector. The sample was irradiated with a 1 kW slide projector (HILUX-HR, Tokyo Master) using glass optical filters (Y47, Toshiba). The difference FTIR spectra shown in this paper are the means of four to eight independent recordings, each of which was the mean of 64 scans (resolution = 2 cm−1). Our recent low temperature spectroscopy results with PYP in 66% glycerol buffer showed that PYPL is formed by the irradiation of PYP at −80 °C, but PYPM was not observed in the thermal reaction (10Imamoto Y. Kataoka M. Tokunaga F. Biochemistry. 1996; 35: 14047-14053Crossref PubMed Scopus (127) Google Scholar). This could be due to the effect of the presence of glycerol at high concentration, because it suppresses the decay of PYPL but accelerates the decay of PYPM(20Meyer T.E. Tollin G. Hazzard J.H. Cusanovich M.A. Biophys. J. 1989; 56: 559-564Abstract Full Text PDF PubMed Scopus (168) Google Scholar). In the method used here, the film sample can be frozen with no increase in turbidity, and the addition of glycerol was not necessary for the low temperature experiments, thus removing impediments to the formation of PYPM. We tested several irradiation conditions above −80 °C to accumulate PYPM without PYPL for FTIR spectroscopy. Upon irradiation of PYP film with >450-nm light at −40 °C, the absorbance at 350 nm was increased (Fig. 1). Because this product had largely blue-shifted absorption spectrum similar to pB (Fig. 1,inset) and directly reverted to PYP (data not shown), it would correspond to pB (called PYPM hereafter). In this difference spectrum, the absorbance at 460 nm was not increased, indicating that only PYPM was formed. Under this irradiation condition, the difference FTIR spectra were recorded with hydrated and deuterated PYP films. The difference spectra before and after irradiation with >450-nm light at −40 °C are the difference between PYPM and PYP (PYPM/PYP spectrum), in which positive and negative peaks are attributed to PYPMand PYP, respectively (Fig. 2). The prominent bands of PYP were observed at 1736, 1560, 1482, 1437, 1301, 1163, 1058, 1041, 983, and 831 cm−1. On the other hand, the intensities of the bands of PYPM were relatively small, but 1175, 1081, and 994 cm−1 bands were characteristic for PYPM. Several bands of them were affected by D2O substitution.Figure 2Difference FTIR spectra between PYP and PYPM in hydrated (solid line) and deuterated (dotted line) samples. The IR spectrum of each sample was recorded before and after irradiation with >450-nm light for 600 s (means of 64 scans at −40 °C). After warming the sample to the room temperature, recording was repeated. The spectra shown were normalized at 1163 cm−1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Recent resonance Raman spectroscopy covering the range 1750–1000 cm−1 (14Kim M. Mathies R.A. Hoff W.D. Hellingwerf K.J. Biochemistry. 1995; 34: 12669-12672Crossref PubMed Scopus (139) Google Scholar) was helpful for the of FTIR It was reported that the prominent vibrational modes of PYP were observed at and cm−1 in H2O buffer (14Kim M. Mathies R.A. Hoff W.D. Hellingwerf K.J. Biochemistry. 1995; 34: 12669-12672Crossref PubMed Scopus (139) Google Scholar) and were cm−1 by D2O It was that the chromophore of PYP is the chromophore bands would be It was that the deprotonated chromophore bands at and cm−1 but protonated there is only one band at cm−1 (14Kim M. Mathies R.A. Hoff W.D. Hellingwerf K.J. Biochemistry. 1995; 34: 12669-12672Crossref PubMed Scopus (139) Google Scholar). The spectrum reported corresponding negative at 1482, 1437, and 1163 cm−1 and positive at 1175 cm−1. The bands at 1482, 1437, and 1163 cm−1 were observed only on the negative indicating that PYPM not have vibrational modes corresponding to the fact that the 1175 cm−1 band was strongly affected by D2O that the chromophore of PYPM is To that the 1175 cm−1 band of PYPM is the vibrational mode of the chromophore, spectrum was recorded using PYP chromophore at under the same irradiation −40 The of 1175 cm−1 band with (Fig. was similar to that with D2O (Fig. 2). Therefore, this band is attributed to the chromophore, which that the chromophore of PYPM is The negative band at 1736 cm−1 be because it could be the C=O stretching mode of the COOH group of acid or A similar band was by FTIR spectroscopy (18Xie A. Hoff W.D. Kroon A.R. Hellingwerf K.J. Biochemistry. 1996; 35: 14671-14678Crossref PubMed Scopus (183) Google the irradiation was different from that of the present The of the negative band without the positive band of the COOH residues of PYP (7Baca M. Borgstahl G.E.O. Boissinot M. Burke P.M. Williams D.R. Slater K.A. Getzoff E.D. Biochemistry. 1994; 33: 14369-14377Crossref PubMed Scopus (291) Google the most for the of this band would be Glu-46 because its OH group directly interacts with the phenolic oxygen of the chromophore and it is inside the protein. To a Glu-46 → Gln mutant (E46Q) of PYP was prepared (19Mihara, K., Hisatomi, O., Imamoto, Y., Kataoka, M., and Tokunaga, F. (1997) J. Biochem. (Tokyo), in press.Google and its vibrational modes were studied by FTIR spectroscopy. the absorption of in the region is from PYP (19Mihara, K., Hisatomi, O., Imamoto, Y., Kataoka, M., and Tokunaga, F. (1997) J. Biochem. (Tokyo), in press.Google the corresponding to PYPM accumulated under the same irradiation conditions −40 (data not FTIR spectroscopy was then using and a spectrum was (Fig. As the spectrum of not a indicating that the C=O stretching mode of the COOH group of Glu-46 is the of this Namely, Glu-46 is protonated in PYP and its proton in the PYPM state. on present the for proton movement during the PYP photocycle is shown in On absorption of a the chromophore of PYP to (15Kort R. Vonk H. Xu X. Hoff W.D. Crielaard W. Hellingwerf K.J. FEBS Lett. 1996; 382: 73-78Crossref PubMed Scopus (204) Google Scholar). are two possible for the isomerization of the one is that the and the other is that the A. Hoff W.D. Kroon A.R. Hellingwerf K.J. Biochemistry. 1996; 35: 14671-14678Crossref PubMed Scopus (183) Google Scholar) proposed that the hydrogen bond between the OH group of Glu-46 and the phenolic oxygen of the chromophore in PYPL and the However, irradiated PYP at to irradiation yields a of PYPB and PYPH (10Imamoto Y. Kataoka M. Tokunaga F. Biochemistry. 1996; 35: 14047-14053Crossref PubMed Scopus (127) Google Scholar, W.D. Van Grondelle R. Hellingwerf K.J. Photochem. Photobiol. 56: Scopus Google so the changes in C=O stretching mode of PYP observed at be attributed to the formed absorbs a and it is or not the photoproduct accumulated at has the Therefore, are to the mechanism of the isomerization of the However, the is likely because the phenolic oxygen interacts with Glu-46 and and it is by the hydrogen bond. In the of retinal the protein moiety has the for the of the chromophore, and the the on Y. T. T. H. J.P. Biochemistry. 1987; 26: PubMed Scopus (48) Google Scholar). in PYP and retinal protein systems, the moiety on of the conformational isomerization would to the and of photoreceptor proteins. The present have demonstrated the first evidence that the phenolic oxygen of the chromophore is protonated in PYPM. The absorption of PYPM is 355 nm W.D. Van Stokkum I.H.M. Van Ramesdonk H.J. Van Brederode M.E. Brouwer A.M. Fitch J.C. Meyer T.E. Van Grondelle R. Hellingwerf K.J. Biophys. J. 1994; 67: 1691-1705Abstract Full Text PDF PubMed Scopus (252) Google which is to that of PYP at neutral (11Meyer T.E. Yakali E. Cusanovich M.A. Tollin G. Biochemistry. 1987; 26: 418-423Crossref PubMed Scopus (287) Google Scholar, A.R. Hoff W.D. J. Crielaard W. Hellingwerf K.J. J. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Because the phenolic oxygen of PYP is protonated, the largely blue-shifted absorption spectrum of PYPM is due to of the However, the that and the nearby positive charge the absorption spectrum of PYPM is not excluded. These be in In retinal the chromophore of the dark is protonated at the of the and It is deprotonated and neutral in the intermediates and In the p-coumaryl chromophore is in PYP but is neutral in PYPM. the in the protein and the of retinal proteins and PYP neutral in intermediates, which are to be formed as the of conformational changes (17Maeda A. Isr. J. Chem. 1995; 35: 387-400Crossref Scopus (133) Google Scholar, 1996; PubMed Scopus Google Scholar). In isomerization of the chromophore is in (15Kort R. Vonk H. Xu X. Hoff W.D. Crielaard W. Hellingwerf K.J. FEBS Lett. 1996; 382: 73-78Crossref PubMed Scopus (204) Google Scholar). These are the events for the protein conformational changes the photoreceptor proteins and would be the most Namely, the chromophore is the of the hydrogen-bonding network that its tertiary structure in the dark state. of the chromophore on photon absorption results in the of its As a the hydrogen-bonding network is and the protein conformational In PYP, the phenolic oxygen of the chromophore interacts with Tyr-42 and and the chromophore is The of this interaction would the structure of the chromophore The present that the 1736 cm−1 band of PYP is the C=O stretching mode of the COOH group of Glu-46. of this mode in PYPM that Glu-46 is protonated in PYP but deprotonated in PYPM. the chromophore of PYPM is This that the a of the chromophore and Glu-46 of PYPM are to those in but that those of PYP are different due to the in the protein. Therefore, the chromophore would be to the in PYPM as the of the protein conformational changes. Because the of the chromophore and Glu-46 are the for proton pathways would be that the proton at Glu-46 is transferred to the chromophore in to Glu-46 in the (Fig. In the chromophore would uptake proton from the or Tyr-42 would as the proton This not the proton release and uptake with the formation and decay of PYPM. Therefore, another amino acid residue be in the proton in movement of the as with T. T. K.J. Biochemistry. PubMed Scopus Google Scholar). FTIR studies on the other intermediates using site-directed the pathway of proton We of Tokyo for the of E. BN We for on the and for

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSynthesis of the cytotoxic germacranolide eucannabinolideW. Clark Still, Shizuaki Murata, Gilbert Revial, and Kazuo YoshiharaCite this: J. Am. Chem. Soc. 1983, 105, 3, 625–627Publication Date (Print):February 1, 1983Publication History Published online1 May 2002Published inissue 1 February 1983https://pubs.acs.org/doi/10.1021/ja00341a055https://doi.org/10.1021/ja00341a055research-articleACS PublicationsRequest reuse permissionsArticle Views968Altmetric-Citations54LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts