917
J. Phys. Chem. 1002, 86,917-921
TABLE VI: Correlation Effects on Molecular Inversion Barriers (in kcal/mol) molecule SCF CI A Ea ref CH,CH,“3
“3
H30+ H,O+ SiH3PH, SH3+ a AE
2.0 1.5 5.6 6.2 2.3 2.0 26.2 34.4 32.8
1.7 1.7 5.2 5.9 1.5
1.3 27.3 36.7 33.6
= E(C1) - E(SCF).
t0.2 -0.2 t0.4 + 0.3 + 0.8 t 0.7 -1.1 -2.3 -0.8
6 12a 12c 4a 12c 12e 12d b b
This work.
techniques. They used the experimental geometry for PH3(pyr) and the same experimental bond distance for PH,(pl) and performed only a partial CI. They also studied the barrier using an MB D STO basis set and found results virtually identical with ours. The best calculations (Basis Set DZ02 of ref 16) are shown in Table V with no polarization functions and with polarization functions only on P(DZ D). The DZ results at the SCF level are very low compared to our standard value and with our DZ results. The DZ + D result at the SCF level is in reasonable agreement with the DZ + P result showing that the polarization functions on H play a smaller role than the polarization functions on the central atom. A similar result has been previously observed for amines.’Ob The trends that we observed at the CI level are reproduced by Scott and Sutcliffe’s calculations; CI increases the barrier at the DZ level and decreases it at the DZ + D level. The effects are, however, much larger than we observed. Comparison of these results using CGTO basis sets with our STO calculations demonstrates the importance of obtaining a properly contracted basis set in order to reproduce calculations using an STO basis. The size of the correlation correction to the inversion barrier using the NHF basis is surprising considering the magnitude of the correlation correction at the DZ P level. Although a 5% correction due to correlation effects is
+
+
+
small, the magnitude, 2.3 kcal/mol, is of chemical importance. This magnitude is larger than what has previously been observed for other hydrides. The barriers and correlation effects in other hydrides are shown in Table VI. Since a large correlation effect was found for PH3, we calculated the inversion barrier in SH3+using the same size basis set as employed in the PH3 calculations.28 The total SCF energies are SH,+(pyr) = -398.99622 au and SH,+(pl) = 398.94274 au, while the SCF-CI energies are SH,+(pyr) = -399.16892 au and SH3+(pl)= -399.11644 au. This yields barriers to inversion of 33.6 and 32.9 kcal/mol at the SCF and SCF-CI levels, respectively. Correcting for the effect of quadrupole excitations yields a barrier of 32.8 kcal/mol for SH3+. Thus, correlation lowers the barrier in SH3+by 0.8 kcal/mol using an NHF basis. This is in contrast to the result found with a DZ P basis where correlation corrections increase the barrier by 0.6 kcal/mol. The correlation corrections show certain trends. For inversion barriers of compounds containing second row ( n = 2) atoms, most calculations show that correlation increases the barrier, with the largest correction being found for H30+. (The exception is the calculation for CH< using SCEP). The barriers for compounds containing third row atoms show the opposite effect with correlation corrections decreasing the size of the inversion barrier. The effect in this row is largest for PHB. In conclusion, we have calculated the inversion barrier in PH3 to be 34.4 kcal/mol and the inversion barrier in SH3+to be 32.8 kcal/mol. A moderate sized correlation effect is observed on the inversion barrier. This correlation effect is shown to be quite basis set dependent and is found only when very large basis sets are employed.
+
Acknowledgment. D. S. Marynick acknowledges the Robert A. Welch Foundation (Grant Y-743) and the Organized Research Fund of the University of Texas at Arlington for partial support of this work. (28) The baais set is taken from ref 22. The geometries are the SCFCI optimized structures obtained with a DZ + P basis given in ref 9.
Electron Spin Resonance Monitorlng of Ligand Ejection Reactions Following Solid-state Reduction of Cobalt Globin and Cobalt Protoporphyrln Complexes L. Charles Dlcklnson’ Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 0 1003
and M. C. R. Symons Department of chemistry, University of Leicester, Leicester, England LE 1 7RH (Received: April 4, 198 1; In Final Form: October 29, 198 1)
Cobaltihemoglobin, isolated a and 6 chains, and cobaltimyoglobin in aqueous solution at neutral pH were irradiated at 77 K with 3 Mrd of @ C ‘o y-rays. These diamagnetic Co(II1) species are converted to paramagnetic Co(I1) species in high yield. The EPR spectra are identical with those of authentic six-coordinate cobalt(I1) porphyrins. Upon partial annealing of the species, the EPR spectrum transforms irreversibly to that of a five-coordinatespecies, indicating that at 77 K these cobaltiglobins are “cobaltichromes”in analogy to the hemichromes of the native iron species. Differences are seen among all of the six-coordinate,reduced protein ligated species. This ejection of the sixth ligand with thermal annealing after addition of one electron to the d,2 orbital of the cobalt porphyrin also occurs in aqueous glasses of cobalt protoporphyrin IX in pyridine, n-butylamine, or quinuclidine. The five-coordinate species in aqueous media are stable with annealing to room temperature.
A number of approaches have been made toward an understanding of the bonding of ligands to hemoglobin. 0022-3654/82/2086-0917$01.25/0
Although X-ray crystallography offers a clear, static picture of geometry and bonding, other techniques contribute to 0 1982 American Chemical Society
918
Dlckinson and Symons
The Journal of Physical Ch8miSfty, Vol. 86, No. 6, 7982
TABLE I: EPR Parameters for Cobalt Porphyrin Complexes Generated b y 7-Irradiation five-coordinate complexesa species
ol(cOHb) P('OHb) a ,P ,(coHb) CoMb COPPIX COPPIX COPPIX COPPIX
ligand
g1 (t0.002)
PYr BuNH, quin MTE
2.325 2.312 2.324 2.328 2.322 2.320 2.319 2.282,b 2.299
six-coordinate species
g II
( i 0 . 0 0 2 ) s9c0A/j,cG ( m k ) 2.029 2.040 2.040 2.046 2.030 2.032 2.028 2.026
78 5 2 ( 7 . 4 ) 78 ?: l ( 7 . 4 ) 75 ( 7 . 1 ) 76 r 0 . 5 ( 7 . 3 ) 81.7 ( 7 . 7 4 ) 80 ( 7 . 5 9 ) 82.5 ( 7 . 8 1 ) 89 ( 8 . 4 1 )
laNA11
gl
59c0A1,cG ( m k )
16.4 (1.55) 15.5 ( 1 . 4 7 ) 16 ( 1 . 5 2 ) 15.9 (1.51) 16.3 ( 1 . 5 4 ) 14 i' l ( 1 . 3 3 ) 17.2 (1.63)
2.202 2.206 2.220 2.226 2.215 2.219 2.211 2.320
80 i 1 ( 8 . 2 ) 79 i l ( 8 . 1 4 ) 71.5 ( 7 . 4 ) 65 ( 6 . 7 ) 61 ( 6 . 3 ) 68(7.0) 62 (6.4) 84 ( 9 . 1 )
a Unresolved CoAl estimated as < 8 G from width o f envelope of perpendicular feature. Parallel feature not detected. clear assignment.
a broader picture of the dynamic chemistry involved. Among the interesting species prepared in pursuit of an understanding of ligand bonding have been cobalt hemoglobin and These species have been particularly amenable to EPR study2 because both the oxy and deoxy species have one unpaired electron per heme unit. This property has allowed measurement of the complete oxygen-17 hyperfine tensor for each atom in oxycobalt my~globin.~ A species isoelectronic with oxycobalt myoglobin has been generated in y-irradiated oxyhemoglobin and oxymyoglobin5 and is identified as a one-electron addition center, Fe02-. The work presented here is the outcome of attempts to generate similar centers in y-irradiated cobalt myoglobins with various ligands. The efforts to understand what is seen in the cobalt proteins are supported by a number of model studies with cobalt porphyrin in a variety of media.
Experimental Section Protoporphyrin M was purchased from Sigma Chemical Co., converted to the cobalt porphyrin as previously described,2 and purified by chromatography on silica geL3 Cobalt myoglobin and cobalt hemoglobin were prepared as previously d e ~ c r i b e d .Subunits ~ were separated according to the procedure of Beychock and were left as the p-mercuribenzoate derivatives.6 Each species was converted to the cobalt(II1) derivative by oxidation with a 2-fold excess of potassium ferricyanide by exposure at room temperature for 30 min followed by use of a Sephadex G-25 column (2 X 30 cm) in 0.01 M pH 7 Naphos buffer. Nitrosylcobalt myoglobin was prepared by exposing deoxycobalt myoglobin to 10 mm of NO in a nitrogen atmosphere. Cobalt(II1) protoporphyrin IX (1 mg) was dissolved in a solution of 0.25 mL of the base, 0.25 mL of ethylene glycol, and 0.50 d of deionized water. Pyridine and n-butylamine were reagent grade and not further purified. Quinuclidine hydrochloride was treated with aqueous NaOH, extracted into ether, and washed three times. 2-(Methylthio)ethanolwas purchased from Aldrich. (1) (a) Hoffman, B. M.; Petering, D. H. h o c . Natl. Acad. Sci. U.S.A. 1970,67,6371.(b) Hsu, G.C.; Spilburg, C. A.; Bull, C.; Hoffman, B. M. J.Biol. Chem. 1972,247,4219. (2)(a) Chien, J. C. W.; Dickinson, L. C. h o c . Natl. Acad. Sci. U.S.A. 1972,69,2783. (b) Abbreviations: EPR,electron paramagnetic resonance; pyr, pyridine;MTJ3,2-(methylthio)ethano1;BuNH2,n-butylamine; CoPPIX, cobalt protoporphyrin M; quin, quinuclidine;Co(p-OCH3)TPP, a,fl,y,6-tetrakis@-methoxyphenyl)porphinatocobalt(II). (3)Yonetani, T.;Yamamoto, H.; Iizuka, T. J. B i d . Chem. 1974,249, 2168 and references therein. (4) Dickinson, L. C.; Chien, J. C. W. R o c . Natl. Acad. Sci. U.S.A. 1980,77,1235. ( 5 ) (a) Symons, M. C. R.; Peteraon, R. L. Biochin. Biophys. Acta 1978, R. SOC.London, 535,241-246.(b) Symons, M.C. R.; Peterson, R. L. BOC. Ser. B 1978,201,285-300. (6)Yip, Y. K.;Waks, M.; Beychok, S. R o c . Natl. Acad. Sci. U.S.A. 1977,74,64.
Weak feature also at 2.320; un-
A
-7
e
?"AL
2Goo
28bO
30b0
'
Flgue 1. (A) Low-fleld part of the Em spectrum of irradiated %b+. The high-field features are obscured by Intense features from organic radials. (B) Sample in Figure 1A after slow annealing to thawing pdnt.
y-Radiation was carried out on a 1.5-Mrd B°Co source for 2 h in silica tubes at 77 K. EPR spectra were obtained on an E109 spectrometer at 77 K with annealing performed in a liquid nitrogen finger Dewar after removal of the liquid nitrogen. Samples were recooled to 77 K whenever significant spectral changes were observed.
Results Cobalt Proteins. Some initial work was done on y-irradiation of &MbNO and &MbO, in frozen glasses. Weak signals were seen to low magnetic field relative to the EPR signal from CoMb02and the organic and peroxy radicals generated. Only implausible mechanisms could be postulated that could lead to species with the observed spectra from the ligated Co(I1) species. Subsequent work with the fully oxidized samples confirmed that these weak signals were due to species formed from impurities of coMb+in the &MbNO and &Mb02. The spectra observed for these samples are found to be identical with the ones discussed below for samples of pure coMb+or coHb+. Figure 1A shows the low-field part of the EPR spectrum of y-irradiated coHb+. The spectrum is seen to have somewhat irregularly spaced hyperfine lines centered near g = 2.220. When the sample was warmed to the point of disappearance of the signal from trapped hydrogen atoms, no change in intensity or spacing of lines was seen. Further warming irreversibly generated the EPR spectrum in Figure 1B. This spectrum has a perpendicular feature at g = 2.324 and a series of parallel cobalt hyperfine lines, each a nitrogen triplet with respective splittings of 76 and 16.4 G. Results nearly identical with those for &Hb+were obtained upon inspection of coMb+samples under similar conditions. As seen from comparison of the parameters in Table I for five-coordinate products from coMbf and CoHb+radiation, no significant differences are seen in the EPR parameters for each heme protein. However, some small parameter differences are seen when the separated
7718Journal of Physical Chernlstty, Vol. 88, No. 6, 1082 919
Llgand Ejectlon Reactlons of Cobalt Complexer
TABLE 11: EPR Parameters for Five-Coordinate Cobalt Porphyrin Complexes Reduced by y. Irradiation and Reduced Chemically
c
I
I
species 811 2.030 COPPIX(pyr) Co@-OCH,)TPP(pyr) 2.026 CoPPIX(BuNH ) 2.032 c ~ ( ~ - o c H , ) T T ~ ( B ~ N H 2.022 ,) 2.028 COPPIX(quin) Co(p-OCH,)TPP(quin) 2.032
coA I/7
G (mk) ref 81.7 (7.74) this work 84.2(7.98)7 80.3 (7.62)this work 82.4 (7.82) 7 82.3 (7.92)this work 84.3 (8.12) 7
all six-coordinated species. The A, values also are diagnostic with A, < 13 G for five-coordinate and >50 G for 2800 2800 3000 six-coordinate species. These assignments to five- and six-coordinationwere based on EPR spectral changes with Flgun 2. (A) Low-field part of EPR spectrum of y-lrradlated CoPPIX variation of concentration and also agree with the theoIn 1:1:2 pyrkilne/ethylene glycol/water at 77 K. (B) Spectrum obtained retical expectations of six-coordination raising the d,z level upon warming of the sample of Flgure 2A to 110 K. and lowering the g anisotropy. Thus, the transition with chains crcoHb and P H b are radiated, though both chains annealing illustrated in both Figures 1 and 2 shows that exhibit the shift from a six- to five-coordinate ligation upon these cobalt-protein and cobalt-base species undergo a warming, yielding spectra comparable to Figure 1. transition from six-coordinationto five-coordination. This Cobalt Porphyrin Complexes. y-Radiation of CoPPIX we term a ligand ejection. It is easy to explain this ejection in aqueous solutions of ethylene glycol was carried out with on the basis of the electronic structure of Co(1II) and Co(II) pyridine, n-butylamine, and quinuclidine. The EPR in the porphyrin macrocycle. Co(II1) has an expected spectra for the pyridine case are given in Figure 2, where ground state of d~,d~,d&.Any ligand, e.g., nitrogen ligand, spectral changes resembling those for the proteins are seen coordinates to the cobalt by donating a pair of electrons when the samples are annealed. In contrast to those of into the empty d,z and/or d+a orbitals. Radiolysis frees the proteins, however, the spectra from the solutions electrons that are subsequently trapped at the acceptor containing the bases switched irreversibly from six- to Co(I1) centers and occupy the d,z orbital. The d,z orbital five-coordination before 110 K was reached in the anis in the direction of the nitrogen nuclei, and the now nealing. This temperature was estimated by the disapoccupied d,z orbital repels the electron pair of the nitrogen. pearance of the signal from hydrogen atoms. EPR paThis system must be fairly delicately poised because it is rameters for all species examined are shown in Table I. possible to force a sixth ligand onto the cobalt in solutions Nitrogen-Sulfur Ligands. A solution that could poat high base concentrations,' and as we see in our results, tentially model cobalt cytochrome c ligation" was prepared the sixth ligand remains on the Co(II1) under the conby adding to 0.75 mL of a cobalt(II1) protoporphyrin IX straint of the rigid low-temperature matrix but is ejected solution in water/ethylene glycol, as above, a stoichiometric upon annealing. This same effect of sixth ligand has been amount of pyridine and 0.25 mL of 2-(methylthio)ethanol found in CO(CN)~"irradiation (ref 8 and references (MTE) (50-fold excess). The EPR spectrum generated by therein) although the ejection occurs readily at 77 K but y-irradiation is indistinguishable from that of Figure 2B, is affected by the lattice. CO(CN)&~has a lower g anindicating the absence of sulfur ligation in this case. Some isotropy and larger hyperfine coupling than the porphyrin water/ethylene glycol solutions of CoPPIX/MTE were case, but the occupied orbitals are the same. irradiated. The parameters for the paramagnetic products It is of interest to establish whether the five-coordinate are given in Table I. species observed after annealing but before thawing the Solvent Effects. Irradiation was performed both in a glass is any different than the five-coordinate species 1:4 pyridine/toluene mixture and in neat pyridine to see generated by chemical reduction in the solution phase and the effects of solvent on the ligand ejection. In the toluthen frozen into the glass. Because no changes were seen ene/pyridine mixture c ~ ~ p P I X ( p ywas r ) ~obtained in high by us upon annealing the five-coordinate species to above yield. The g, and &A, values were identical with those the glass point and then refreezing to 77 K, there can have obtained in the aqueous glycol glasses. After the sample been no differences in the five-coordinate species due to was annealed, the EPR signal gradually weakened, with constraints from frozen solvent or immediate ligands at no evidence of formation of the five-coordinate complex. low temperature. That these species do not differ sigAfter the sample was thawed and refrozen, the EPR signal nificantly from those generated at room temperature by completely disappeared. other means can be seen from Table 11,where our results In neat pyridine, the six-coordinate C O " P P I X ( ~ ~ ~ )are ~ compared with those for C O ( ~ - O C H J T P P .The ~~ complex was not observed in the EPR on irradiation of the hyperfine values we remarkably close, and the &A,,values sample. Instead, an irregular peak centered at g = 2.315 follow the same sequence for the series of ligands for both with about a 100-G line width was observed. This specthe CoPPIX and Co(p-OCH3)TPP cases, Le., BuNHz < trum was also lost on thawing of the sample. pyr < quin. The EPR spectra of the products of irradiation of Discussion aqueous solutions of CoPPM and MTE indicate that there The spectra shown in Figure 1, parts A and B, can be are differences between these species and the species unambiguously ascribed to six- and five-coordinated generated by chemical reduction in neat MTE. coAllfor CoPPIX, respectively, by comparison with the extensive the annealed aqueous solution is 89 G whereas the value study of cobalt tetraphenylporphyrins by Walker.' In for the chemically reduced species is 84 G.12 There is no Table I of that reference it is seen that g, > 2.3 for all apparent reason why these should be different species, and five-coordinated cobalt porphyrin species and g, < 2.3 for I
I
I
I
I
(7) Walker, F. A. J. Am. Chem. SOC.1970,92,4236.
(8) Symons, M. C. R Wilkinson, J. G . J. Chem. SOC.A 1971, 2069.
020
The Journal of Physical Chemistry, Vol. 86, No. 6, 1982
the difference may reflect a solvent influence. This influence would be great for a five-coordinated species because the d , ~orbital is exposed. The six-coordinate species are of particular interest, especially for the protein-CoPPIX systems, because they cannot be detected by EPR spectrometry in the solutionreduced regime. The fifth ligand is undoubtedly the nitrogen of the F8 histidine in the hemoglobins and myoglobin. An obvious possibility for the sixth ligand is the E7-distal histidine, which is held by the protein structure for the ferrous and ferric cases at some nonbonding distance from the iron atom. There is also a possibility, of course, that another protein side chain or water is the sixth ligand. These seem unlikely because there are no other polar nitrogen or oxygen ligands in the heme pocket. Water seems an unlikely possibility because it would create a much weaker ligand field than nitrogen ligands. Our EPR parameters agree closely with those of the known six-nitrogen coordination cases,' and thus it seems that we have both the E7 and F8 imidazole nitrogens bonded to CoPPIX in the protein cases examined. This result gives direct spectroscopic evidence that coHb+ is a "hemichrome". This has been indirectly inferred from titration data that showed, in contrast to FeHb+,no ionizable water coordinated to the metal and two metalprotein bonds. &Hb+ also does not coordinate such species as N3- and CN-.g This difference of coHb+ having two internal nitrogen ligands and FeHb+having one nitrogen and one water ligand is of considerable interest. That six-coordination is possible in these proteins implies sufficient flexibility in the structure for the E7 imidazole N, nitrogen to come in close enough to the cobalt ion to form a bond. In oxymyoglobin recent X-ray crystallographic results at 1.6-A resolution at -12 OCl0 show that the residues in the heme pocket have low thermal motion factors but that the E7 enitrogen has a much larger thermal motion, though not sufficient to place the nitrogen in bonding range for the iron. Thus it appears that CoMb must have a significant conformational difference compared to "Mb or FeMbOz.These differences are probably related to the small changes observed in comparison of Mb02and Mb,lowhich are similar to but of a smaller scale than the R to T transition changes seen in hemoglobins. As the Co-N (F8) bond is shorter than the corresponding Fe-N bond and shows less dramatic changes in distance upon oxygenation one might expect less dramatic effects in the protein to be seen in the cobalt case. Nonetheless, our results do suggest that there are small differences in conformation that can be transmittedfrom the Co-N bond with change of coordination and that changes in coordination may result from accessible conformation states. An understanding of why CoMbis doubly coordinated to the protein and "Mb is singly coordinated to the protein can also be approached from the point of view of affinity. Some insight can be gained by examining the considerable literature on the radiolysis of the Fe(CN),* and Co(CN),* species. The former gives high yields of Fe(CN)64-with only small loss of cyanide ligand.13 The latter shows primarily cleavage of a CN group.14 While this implies, as has been seen with the CoPPIX system, that low-spin cobalt systems easily eject a sixth ligand, it does not ad~
~~~
(9) Risdale, S.;Cassatt, J. C.; Steinhardt, J. J.Biol. Chem. 1973,248, 771. (IO)Phillips, S. E. V. J.Mol. Biol. 1980, 142, 531. (11) McGarvey, B. R. Can. J. Chem. 1975,53, 2498. (12) Dickinson, L. C.; Chien, J. C. W. Znorg. Chem. 1976, 15, 1111. (13) Zehavi, D.; &bani, J. J. Phys. Chem. 1972, 76, 3703. (14)Furuta, N.;Watanabe, T.; Fujiwara, S. Bull. Chem. SOC.J p n . 1976, 49, 1740.
Dickinson and Symons
dress the problem of the relative affinity of the Fe(II1) and Co(II1) systems for the sixth ligand. There appears to be no directly comparable stability constant data for FePPM and CoPPIX for a sixth ligand. However, the pK for Fe(CN)63-is 31 and that for C O ( C N ) ~is~64,15 indicating that while both five-coordinate species have a very high affiity for a sixth ligand, the affinity of the Co(II1) species is larger than that of the Fe(II1) species. Thus, stronger attraction of the sixth ligand to Co"'PP1X as compared to FeII'PPIX may be a contributing factor of the former in binding two endogenous ligands while the latter binds only one. However, it is known that the metal-nitrogen bond length is shorter for Co than for Fe in the five-ligand case. This could cause trans effects or indirect proteinmediated effects so that the causes of the cobalt hemichrome are not separable at the current state of knowledge. Because we see a variation in A , for the six-coordinate Co(I1) protein cases, we believe that the protein-mediated effects are a considerable influence on the formation of the hemichrome. This is also substantiated by the ejection of the sixth ligand at a higher temperature in the proteinligand cases as compared to the CoPPIX-base complexes. I t is unfortunate that the differences among the EPR parameters for the several protein matrices for CoPPIX cannot be interpreted without &A, for the five-coordinate species or &Allor gll for the six-coordinate species. These species are both likely to be of distorted geometries based on the detailed atomic positions of Phillips'O and FeMbOz. It should be noted that the range in &Al is quite large for the six-coordinate species and that there is a change in upon combining a and fi chains. This parameter seems especially sensitive to what must be small distortions in the heme pocket geometry. The case of CO(CN)~~has been studied to show that the d,l orbital occupancy changes Ab and second-orders and third-order"J2 perturbation theory is required to extract meaningful orbital coefficients from the parameters. The CoPPIX-base cases also show little variation in the parameters for n = 1,but for n = 2 the BuNH2case shows both a larger g, and A , than are seen with the other bases. This reflects differences in the heterocyclic vs. aliphatic amines as has been seen by others.' Attempt To Produce CoPPIX Sulfur-Nitrogen Coordination. The sulfur ligand (methy1thio)ethanol (MTE) was of interest because of the sulfur ligation of methionine in cytochrome c.12 Previous attempts to generate a complex of CoPPIX(pyr)(MTE) by reduction of CoPPIX solution containing both ligands in a range of stoichiometries resulted only in complexes with one or two pyr ligands and no MTE. In this work we examined the ligation of these species to ComPPIX by the irradiation method described above. As reduction to Co(I1) was carried out in frozen solution at 77 K any ligand exchange is quenched and we can observe the EPR spectrum of the complex with the ligands of the original Co(II1) complex. However, even in the presence of a 50-fold excess of MTE with a stoichiometric mixture of CoPPIX and pyridine, irradiation results only in a five-coordinate complex with pyridine as the fifth ligand. This further emphasizes the uniqueness of the coordination in cobalt cytochrome c in that nitrogen-cobalt-suifur ligation can be achieved only under the entropic influence of the protein. In solution neither ConPPIX nor Co"'PPIX forms a nitrogen-sulfur ligation. As no five-coordination species was seen in the toluene glass, it appears that the solvent has a strong influence on Co"PPIX(pyr),. Either toluene reduces the stability of (15) Sillen, L. G.; Martell, A. E. "Stability Constants of Metal Ion Complexes",Chemical Society: London, 1964.
92 1
J. Phys. Chem. 1982, 86, 921-927
the CollPPIX(pyr)l to oxidation and the five-coordinate species rapidly reverts to a Co(II1) species or the ability of ConPPIX to eject the sixth ligand is ifiipaired. However, the oxidation to Co(1II) in either case requires an adequate donor. The toluene used was not redistilled and could have contained such donors. The results in neat pyridine are puzzling, particularly in that the main feature of the spectrum closely resembles
the strongest features of the spectrum of the five-coordinate species.
Acknowledgment. This work was supported by a grant from the North Atlantic Treaty Organization. We are grateful to Lorraine Harris for performing some of the experiments and to Tom Provost for preparing the separated hemoglobin chains.
Spectroscopy of Polyenes. 6. Absorption and Emission Spectral Properties of Linear Polyenals of the Series CH,-(CH=CH),-CHO ParRosh K. Das' Radiatlon Laboratory, Unlverstly of Notre Dame, Notre Dame, Indiana 46556
and Ralph S. Becker Lbpartment of Chemlshy, Unh'erstly of Houston, Houston, Texas 77004 (Received: June 8, 198 I; In Final Form: October 8, 198 I)
The results of a detailed study are presented concerning the absorption and emission spectral properties of four homologous polyenals of the series, CH3-(CH=CH),-CHO, n = 2-5, under various conditions of solvent and temperature. A total of four different band systems have been recognized in the well-resolvedabsorption spectra. these have been assigned as arising from singlet K* n (very weak), lBU* lA, (very strong), lAg*+ lA, (cis peak, moderately weak), and lA,*lA, (weak) transitions. Except for the lAg*+ state which is always located at a relatively high energy, the relative location of the remaining three states, viz., lB,*, l$*-, and '(n,r*), is seen to be a sensitive function of polyene chain length and appears to be the most important factor in determining the radiative behavior of the polyenals. Thus, the C6 and C8 aldehydes (n = 2 and 3), with a '(n,?r*)state seen as the lowest singlet state in absorption,do not fluoresce under any condition of solvents (polar and nonpolar) and temperature (296-77 K). The Clz aldehyde ( n = 5) has the dipole-forbidden lAg*state as the lowest singlet state and exhibits fluorescence (at 77 K) that is moderately strong is not dependent on excitation wavelength). In the case of the intermediate polyenal, Cloaldehyde (n = 4),the three low-lying states, viz., l(n,?r*),lB,*, and l$*-, appear to be nearly degenerate based on their locations in the absorption spectra. While no fluorescence is observed for Clo aldehyde in 3-methylpentane at 77 K, it fluoresces very weakly in 2-methyltetrahydrofuranat 77 K where C$F is practically independent of excitation wavelengths; moreover, fluorescence is moderately strong in EPA at 77 K where h is very strongly dependent on excitation wavelengths. No phosphorescence is observed for any of the polyenals under study.
-
-
Introduction Because of their importance in absorbing and transducing light energy through various photobiological processes, polyenes have always been subjects of considerable interest for research concerning their spectroscopy and photodynamics. The relatively recent finding~l-~ that in polyenes there exists a dipole-forbidden singlet excited state of primarily l$* character near or below the strongly allowed lB,* state has generated a great deal of renewed vigor in the theoretical and experimental work regarding the excited-state properties of polyenes. In polyene al(1)Hudeon, B. S.;Kohler, B. E. Chem. Phys. Lett. 1972,14,299-304. J. Chem. Phys. 1973,59,4994-5002. (2)Christensen, R.L.;Kohler, B. E. J. Chem. Phys. 1975,63,1837-46, J.Phys. Chem. 1976,80,2197-2200. (3)Schulten, K.;Karplus, M. Chem. Phys. Lett. 1972, 14, 305-9. Schulten, K.;Ohmine, I.; Karplus, M. J. Chem. Phys. 1976,64,4422-41.
Ohmine, I.; Karplus, M.; Schulten, K. Ibid. 1978,68,2298-318. (4)Becker, R.S.;Hug, G.; Das, P. K.; Schaffer, A. M.; Takemura, T.; Yamamoto,N.; Waddell, W. J. Phys. Chem. 1976,80,2265-73,and references therein. (5) Takemura,T.;Das, P. K.; Hug, G.; Becker, R. S. J.Am. Chem. SOC. 1976,98,7099-101,1978,100,2626-30,Takemura, T.;Hug, G.; Das, P. K.; Becker, R. S. Ibrd. 1978,100,2631-4. (6) Song, P. S.; Chae, Q.; Fujita, M.; Baba, H. J. Am. Chem. SOC. 1976, 98.ai9-24.
0022-385418212086-092 1$0 1.2510
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dehydes," there is an additional low-lying singlet excited state, viz., '(n,a*), that plays a very important role in determining the spectral and photodynamical behavior of these polyene systems. The notable dependence of the relative location of the three low-lying singlet excited states, namely, lB,*, 'Ag*-, and '(n,?r*), on polyene chain length and various structural factors and environmental conditions makes the photophysics, photochemistry, and spectroscopy of the polyene aldehydes complex as well as interesting. In the previous papers7+ of this series we have reported on the spectral and photophysical properties of polyene systems ending with various functional groups and related to retinyl polyenes as homologues. One interesting feature of the spectroscopy of retinyl polyenes and their homologues (particularly the shorter ones) is that their spectra, both absorption and emission, are generally broad and unresolvable even in a low-temperature glass matrix. This has been s h ~ w n ~toJ ~ be' related, ~ in one way or other, (7)Das, P.K.;Becker, R. S. J. Phys. Chern. 1978,82,2081-93.1978, 82,2093-105. (8)Das, P.K.;Kogan, G.; Becker, R. S. Photochern. Photobiol. 1979, 30,689-95. (9)Becker, R.S.;Das, P.K. J. Phys. Chern. 1980,84,2300-5.Photochem. Photobiol. 1980,32,739-48.
0 1982 American Chemical Society
