BIOINORGANIC
CHEMISTRY 4,99-108 (1975)
99
A Rudy of the Temperature Dependence of the Electronic Spectra of Alkyl Cobaloximes A. BLANCO-LABRA,* A. CARTANO, M. CHU,** and L. L. INGRAHAM University of Gzlifornia. Davis, Department of Biochemistry and Biophysics, Gzlifomia 95616
AB!STRAfl The temperaturedependence of the electronic spectra of the alkyl-pyridinatocobaloximes iu benzene solution was found to be due to an equilibrium existing between two forms proposed to be inner-sphere and outer-sphere coordination compounds_ Evidence is presented to rule out the possrbiiitiesof equilibria with pentacoordinated or dimeric forms, as well as a form with the cobalt out of the plane of the dimethylglyoxime.
INTRODUCTION It is well known that the electronic spectra of vitamin E312 and related compounds are temperature dependent. Isosbestic points [ 1, 2, 33 occur which indicates that the higher temperature form is not merely the result of a decreased hgand field on the electronic structure of the Iower temperature form but rather an equilibrium existing between two spectroscopic forms. These two forms are very rapidly interconverted; the half life is < 20 psec 13 1_ The existence of equilibrium between two electronic forms has also been supported by nmr studies [4], showing reversible changes of H-10 of the con-in ring on varying temperature, and by circular dichroism studies [S] _ The circular dichroisrn is of particular interest because it shows an inversion of the spectra. This can also be brought about by &and substitution. A possible explanation is that these changes are a result of conformational changes in the coenzyme. Firth et al. [2] refer to the conformational change as the result of an equilibrium between pentacoordinated and hexacoordinated forms, in which the form at the higher energy would be the one with an axial ligand completely removed from the cobalt atom. *Present address: Department0 de Bioquimica, Divisi6n de Estudios Superiores Facultad de Quiiuica, UniversidadNational Autonoma de M&ico, MZdxico20, D.F. **Present address: Department of Chemistry, Bloomsburg State College, Bloomsburg, Pennsylvania17815. 0 American ElsevierPublishingCompany, Inc., 1975
A. BLANCO-LABRA,
100 This paper describes a study of the temperature spectra of a simpler system, the alkyl-cobaloximes
et al.
dependence of the electronic in benzene-pyridine solution_
The cobaloximes are of special interest because of their parallelism cobalamins in chemical and physical properties [ 61.
to the
EXPERIMENTAL Materials used in the synthesis of compounds were all reagent grade. Liquid pyridine derivatives were distilled before use. Solvents used for the spectral studies were all spectral quality. Ultraviolet and visible absorption spectra were obtained by using a Gary Model 14 recording spectrophotometer. In the thermal difference study, scanning was with a slide wire for the O-O-O.1 absorbance range and each compartment was thermostated separately_ Solutions were all 2.5 X IOm3 &I (unless specified otherwise) and a minimum of 25 mm was allowed for each sampie to reach the desired temperature before the spectra were taken. Methyl, ethyl, isopropyl-pyridinato-cobaloximes as well as methyl-aquocobaioximes were all prepared by the method reported by Schrauzer et al. [ 7]_ Attempts to obtain the tert-butyl derivative failed even when the Grignard method was used_ The purity of all compounds were checked by ultraviolet, visible, and nmr spectroscopy_ Spectral quality solvents were used to prepare the solutions_
RESULTS
AND DISCUSSION
The electronic spectrum of methyl pyridinato cobaloxime in benzene solution was found to be temperature dependent (Fig. 1) as found previously for the cobalamin derivatives_ The temperature dependence of the electronic spectra of the cobaloximes could be the result of several factors. These include (a) a shift in the spectrum as a result of longer bond distance from increased vibrations and hence a lower ligand field at higher temperatures, (b) dimer formation with the elimination of the pyridine ligands, (c) ligand exchange. (d) equilibrium between hexacoordinated and pentacoordinated forms, (e) nonplanar cobalt, or(f) inner and outer pyridinato complexes_ The presence of isosbestic points in the electronic difference spectra of alkyl cobaloximes at various temperature differences in Fig. 1 indicates two separate spectral species and immediately rules out the first possibility because a continuously decreased ligand field with temperature would cause a continuous shift in the spectrumThe dimer of methyl cobaloxime is known (81 and is a possible explanation for the temperature-dependent spectrum in benzene_ Dimer formation, &and exchange (which must be impossible in benzene solution), and an equilibrium between hexacoordinate and pentacoordinate forms ah require the loss of a
pyridine molecule. To determine whether an equilibrium occurs in which the pyridine is lost, temperature-difference spectra were taken of methyl-pyridinato-cobaloxime in
ELECTRONIC SPECTRA OF ALKYL COBALOXIMES
101
FIG. 1. The temperature difference spectra of 2.5 X 10-3M methyl-pyridinato-cobalosime. The solvent was benzene which was deaented with nitrogen. The temperatures in each cell was 1” and Z’, lo and 7O, 1” and 12”, 1” and 15”, 1” and 20°, 1” and 29, 1” and 30° as we proceed from the smallest to the largest deviations_ The ordinate is a linear optical density scale and the abscissa is a linear waveIen$h scale.
benzene containing increasing pyridine concentrations from 1% to 90%. The experimenta data (Fig. 2) show no changes with pyridine concentration. These results ehminate the possibility of an equilibrium with either a pentacoordinated There are spectral differences between primary and secondor dimeric forms. ary alkyl cobalamins as a result of a change in position of the cobalt atom. [9] _ Cobalt(i)cobinamide can be alhylated by a bulky substituent only when a strong ligand in the sixth position is absent. Accordin g to Brodie this would allow the cobalt to remain above the pIane of the corrin system_ ConverseIy, the presence
A. BLANCO-LABRA, et ai.
III
WAVELENGfH (nm) I
I
I
I
I
I
I
I
I
I,
32@340360380400420440460480Mo520540660580600
I,,
FIG. 2. The temperaturedifference spectra of 0.0005 methyl-pyridinato-cobaioximein benzene-pyridinesolutions at temperaturesof 10°C vs 25°C. Dotted line is the difference spectrum in a solution containing 1% pyridine, the solid line in 40% py-ridine and the dashed line in 90% pyridine. The ordinateis a linearoptical density scaleand the abscissais a linear wavelengthscale.
of a strong ligand (e.g., 5,6-dimethylbenzimidazol or pyrimidine) would tend to force the cobaIt into the plane of the corrin, and this precludes the formation of a carbon-cobalt bond with a bulky allcyl group. Brodie [9] did not observe these two forms in equilibrium in the same compound but such an equilibrium between two positions for the cobalt atom would be another possible explanation for the temperature-dependent
the cobaloximes. In order to test this as a possible explanation spectrum, the Emax (445468
spectra of
for the temperature-dependent
nm) of the temperature-difference
spectra were
compared for the methyl, ethyl, and isopropyl derivatives expecting that if the two existing forms in the equilibrium correspond to the forms with the cobalt in the plane of the ring and out of the plane. then it would be expected that the proportion of the high-ener,~ form (out of the plane) would greatly increase in the series; methyl, ethyl, and isopropyl as steric hindrance between the alkyl group and the cobaloxime plane is increased_ However, no trend of this type was found (ef_, Fig- 3). These results indicate that the temperature dependent spectra is not the result of the existence of conformational isomers with the cobalt atom in and out of the plane. The only logical explanation remaining is that the temperature-difference spectrum for the alkyl-pyridinatocobaloximes in benzene solution is the result
ELECXRONIC SPECTRA OF ALKYL COBALOXIMES
103
difference spectra (2.5 x lo-” M benzene solution deaerated with nitrogen) methyl (max 390,455) c.45 = 323. isopropylpyridinato-cobaloxime (max 395,456) eq5 s = 28, temperature 30” and 10“. The ordinate is a linear optical density scale FIG. 3. The temperature
and the abscissa is a linear wavelength
scale.
of an equilibrium between an inner complex with a short cobalt-pyridine bond and an outer complex with a long cobalt-pyridine bond as suggested by Hill et al. [ 101 for the cobalamins. The original definitions of inner and outer complexes were in terms of orbital hybridization [ II] _ An inner complex was considered to be bonded by hybrid orbitals formed from d-orbitak with s- and p-orbitals of a higher she11 whereas an outer complex was considered to be bonded by hybrid d-,s-,and p-orbitals ail in
104
-4. BLANCO-LABRA,
et al.
the same shell. Other authors [I21 use the terms “ionic” ad “covalent” as synonomous with inner and outer complexes. The long cobalt-pyridine bond must certainly be less covalent and probably would utilize higher orbitals but could not in the usual sense be considered to be ionic although small charge separations wouId contribute to stability in benzene solution_ An exact description would require a molecular orbital calculation. Equilibrium between inner and outer complexes are known for other compounds and in particular for nickel complexes [ 131. The direction of the spectral shifts between inner and outer complexes is in agreement with our knowledge [ 141 of the electronic structure of the cobaloximes. it is well known that the primary effect of the axial ligand is to lower the energy of the highest occupied molecular orbital. Therefore, all transitions from the highest occupied moIecular orbital would be shifted to a lower energy when weakly interacting axial ligands are replaced by strongly interacting ones as is observed in the temperature-difference spectra. The difference spectra of methyl-aquo-cobaloxime and methyl-pyridinatocobaloxime (Fig_ 4) shows a marked similarity to the temperature-difference spectra of the methyl-pyridinato-cobaloxime. The difference between the aquo and pyridinato derivatives is probably similar to the difference between an inner and outer complex for pyridine because the aquo complex would be weakly held with little x-bonding as in an outer pyridinato complex. Of interest is the observation that the aquo complex also exists as an equilibrium between tier and outer complexes_ In Fig_ 5 are shown the temperature-difference spectra of methyl-aquo-cobaloxime in aqueous solution_ In order to make sure that the temperature-difference spectrum is not caused by conformational changes or ionizations in the hydrogen bridges between the dimethyl glyoxime ligands the cyclic oxime boronic ester of methyl-pyridinato-
FIG. I The concentration difference spectrum of I ml of a satrmated aqueous solution of methyl-pyridinato-cobaloxime each in a l-ml and a S-ml cuvette with the 5-ml cuvette diluted to capacity with water_
ELECTRONIC
SPECTRA
FIG. 5. The temperature
OF ALKYL
COBALOXIhlES
difference spectra of 2.5 X lo-’ M methyl-aquocobaloxime solvent was water deaerated with nitrogen. The temperature was IO” and 30°C. ordinate is a linear optical density scaIe and the abscissa is a linear wavelenth scale.
105
the The
cobaloxime was prepared by treatment with boron trifluoride Cl51 _ This compound also exhibited a temperature-difference spectra. The spectrum shown in Fig- 6 was identical for solutions of composition between 70% pyridine-30% benzene (v/v) to 100% pyridine. Below 70% pyridine the compound became too insoluble for good spectra to be obtained_ In order to estimate the amount of the outer sphere complex, the equilibrium between methyl-pyridinato-cobaloxime and methyl-methanolato-cobaloxime was measured in methanoI. Concentration difference spectra were measured by using 1 cm and 5 cm path length cuvettes with the same amount of methyl-pyridinato-cobaloxime in each cuvette but fiied to capacity with methanol_ Again the difference spectra
A. BLANCO-LABRA,
106
I1
360
IllI
400
1
I,‘,
440 480 WAVELENGTH
et ai.
I,
520
560
600
(nm)
FIG. 6. The temperature difference spectrum of 5 X 10m4Mboron fluoride etherate of methyl-pyridinato-cobaioximein 80% pyridine-20% benzene solution (v/v)_ The temperatureswere 9.8”C and 20°C. appears
very similar to the temperature-difference
spectra. The concentration
dependence of the peak at 445 nm in methanol is shown in Table 1. This data treated by the method of Viale and KalIen [ 161 gives an equilibrium constant of 2,740 t 5% for the reaction of methyl-methanolato-cobaloxime with pyridine to form methyI-pyridinato-cobaloxime_ The value of this equilibrium combined with a knowledge of the AOD between the two cells allowed us to calculate a value of the difference in extinction coefficients for the methanolato and pyridinato complexes of approximately 760. if we assume this same difference in extinction coefficient between the tier and outer complexes of pyridine and we neglect
the amount
this temperature
of outer complex at 1OC (the extinction coefficient at is assumed to be that of the inner complex), we can estimate
the amount of outer complex form shown in Table 2. The actual amount of outer form would be increased over these values by any outer form at i°C_ From the temperature dependence of the amount of outer form, the &!Z for the difference between outer and inner complexes can be estimated to be about 8.8 kml per mole. DISCUSSION
We do not intend to extrapolate these results to explain all the temperaturedependent
spectra observed for the cobalamins aithough the behavior of the
107
ELECTRONIC SPECTRA OF ALKYL COBALOXIMES TABLE
1
The Absorption Differences at 445 nm of I ml of Methyl-Pyridinato-Cobaloxime in Methanol at the Concentration Listed in Both a l-ml and a S-ml Cuvette with the 5-ml Cuvette Diluted to Capacity with Methanol_ Concentration
of
(X 104)
A Absorbance 0.360 0.123 0.048 0.032 0.0195 0.0085
14.5 5.83 2.91 2.33 1.75 1.16
TABLE 2 The Equilibrium Constant for the Formation of the Outer Complex Form of Methyl-Pyridinato-cobaIoxime and the Percent of Material in the Outer Complex Form at Various Temperatures in Benzene Solution.
AOD
T°K (vs T1 = l°C)
(445 nm X 104)
275 280 285 288 293 298 303
46 73 88 112 154 185 242
KeqX IO3
c/oOuter form
2.4 3.6 4.7 5.9 8.2 9.8 12.9
0.24 O-38 0.46 0.60 0.81 0.97 1.27
temperature-difference spectra and the similarity of the high-temperature form to the aquo derivative [ l] is analogous to the results reported here. There are probably several explanations depending on the conditions for the temperaturedependent spectra of the cobalamins. However, alkyl-pyridinato-cobaloximes in benzene solution appear to exist in a temperature-dependent equiiibrium between inner and outer forms. The outer form is in low concentration relative to the inner complex form. Exchanges of the fifth ligand are likely to be very important for many functions of vitamin B 1Z [ 171. An example is the proposed mechanism for methane production [ 181, which first requires exchange with a mercaptan before cleavage of the carbon-cobalt bond. Exchange reactions at the 5th ligand position in methyl-aquo-cnbaloxime is 25.6 kcal; a much larger value than our estimate for the formation of an outer complex. Therefore, we visualize
108
A. BLANC&LABRA.
et al.
a rapid reversible equihbrium between the inner and outer complexes followed by a rate-determining step for dissociation of the outer complex_ Because the outer complex is a considerably higher energy than the inner complex, the outer complex wilI dissociate faster than the inner complex_ If the enzyme were capable
of holding
the fifth
ligand
in an outer
complex,
the exchange
reactions
would be very much more rapid. If true, this would be another example of an entatic state of a metallo enzyme [20] in which the strain produced by the
protein increased the reactivity of the active penter. We note that spectral changes We 08285
are observed
wish
to
when cobalamins are bound to an enzyme
acknowledge
and a fellowship
from
the
aid
of
COivACYT
U.S. {from
Public Mexico)
Health
[ 10) _ Service
Grant
GM
for A-B-L_
REFERENCES I_ J_ Fo_x, R_ Banninger, R. Draper and L. L_ ingraham, Arch_ Biochem Biophys- 125, 1022 (1965)_ 2. R_ A_ Firth, H- A. 0. Hill, B. E_ Maan. J_ $1. Pratt, R_ G. Thorp, and R. J. P_ Williams,J.
them_ Sot_ (A), 2419 (1968). 3_ T_ Darwin,J. Am. Chem. Sot. 93,2629 (1971). 4. H. A. 0. Hill, B. E. Mann, J. M. Pratt and R. J. P. Williams, J. Chem. Sot. (A), 564 (1968). 5. R. A. Firth, H. A. 0. Hill, J. M. Pratt, R. J. P. Williams and W. R. Jackson, Biochemistry, 6,2178 (1967)_ 6_ G. N_ Scfuauzer, Act. Chem_ Res. 1,97 (1968). 7. G. N. Schrauzer, L. l? Lee and J. iti Sibert, Act. Chem. Res. 92.2997 (1970). 8. G. N. Schrauzer and R. J_ Windgassen,J_ Am Chem. Sot_ 88,3738 (1966). 9. J. D. Brodie. &cc_ Nat Acad. Sci. 62,461 (1969). lo_ H_ A_ 0. Hill. J_ M. Pratt and R_ J. P_ Williams, Chem Brit. 156 (1969). 1I. H. Taube, them. Rev_ 50,69 (19.52). 12_ F_ H_ Burstailand R. S. Nyho1m.J. Chem. Sot, 3570 (1952). 1K H. Brintzinger and G. C. Hammes, Inorg. Uzem. 5,1286 (1966). 14. G. N. Schrauzer, Natwwfisenshaften 53,459 (1966). 15. G. N. S&rawer and R. J. Windgassen,J. Am. Chem. Sot_ 88,3738 (1966). 16. 17_ 18. 19. 20_
R. 0. Viile and R_ G. Kallen, Arch_ Biochem. Biobhys. 146.271 (1971) G_ Schnauzer et al_. Bioinorg. Chem 2 (2), 93 (1973). J. W_ Sibert and G. N. Schrauzer,J. Am Chem Sot. 92,142l (1971). K. Brown and R. G. Alien, J. Am Chem Sot. 94, 1894 (1972). B. L_ VaJlee and 2_ J_ P_ WilJiams,~oc_ Nat AC&_ Sci 59,498 (1968).
Received lriay 24. I 9 74; revised July I, I 9 74
CHEMISTRY 4,99-108 (1975)
99
A Rudy of the Temperature Dependence of the Electronic Spectra of Alkyl Cobaloximes A. BLANCO-LABRA,* A. CARTANO, M. CHU,** and L. L. INGRAHAM University of Gzlifornia. Davis, Department of Biochemistry and Biophysics, Gzlifomia 95616
AB!STRAfl The temperaturedependence of the electronic spectra of the alkyl-pyridinatocobaloximes iu benzene solution was found to be due to an equilibrium existing between two forms proposed to be inner-sphere and outer-sphere coordination compounds_ Evidence is presented to rule out the possrbiiitiesof equilibria with pentacoordinated or dimeric forms, as well as a form with the cobalt out of the plane of the dimethylglyoxime.
INTRODUCTION It is well known that the electronic spectra of vitamin E312 and related compounds are temperature dependent. Isosbestic points [ 1, 2, 33 occur which indicates that the higher temperature form is not merely the result of a decreased hgand field on the electronic structure of the Iower temperature form but rather an equilibrium existing between two spectroscopic forms. These two forms are very rapidly interconverted; the half life is < 20 psec 13 1_ The existence of equilibrium between two electronic forms has also been supported by nmr studies [4], showing reversible changes of H-10 of the con-in ring on varying temperature, and by circular dichroism studies [S] _ The circular dichroisrn is of particular interest because it shows an inversion of the spectra. This can also be brought about by &and substitution. A possible explanation is that these changes are a result of conformational changes in the coenzyme. Firth et al. [2] refer to the conformational change as the result of an equilibrium between pentacoordinated and hexacoordinated forms, in which the form at the higher energy would be the one with an axial ligand completely removed from the cobalt atom. *Present address: Department0 de Bioquimica, Divisi6n de Estudios Superiores Facultad de Quiiuica, UniversidadNational Autonoma de M&ico, MZdxico20, D.F. **Present address: Department of Chemistry, Bloomsburg State College, Bloomsburg, Pennsylvania17815. 0 American ElsevierPublishingCompany, Inc., 1975
A. BLANCO-LABRA,
100 This paper describes a study of the temperature spectra of a simpler system, the alkyl-cobaloximes
et al.
dependence of the electronic in benzene-pyridine solution_
The cobaloximes are of special interest because of their parallelism cobalamins in chemical and physical properties [ 61.
to the
EXPERIMENTAL Materials used in the synthesis of compounds were all reagent grade. Liquid pyridine derivatives were distilled before use. Solvents used for the spectral studies were all spectral quality. Ultraviolet and visible absorption spectra were obtained by using a Gary Model 14 recording spectrophotometer. In the thermal difference study, scanning was with a slide wire for the O-O-O.1 absorbance range and each compartment was thermostated separately_ Solutions were all 2.5 X IOm3 &I (unless specified otherwise) and a minimum of 25 mm was allowed for each sampie to reach the desired temperature before the spectra were taken. Methyl, ethyl, isopropyl-pyridinato-cobaloximes as well as methyl-aquocobaioximes were all prepared by the method reported by Schrauzer et al. [ 7]_ Attempts to obtain the tert-butyl derivative failed even when the Grignard method was used_ The purity of all compounds were checked by ultraviolet, visible, and nmr spectroscopy_ Spectral quality solvents were used to prepare the solutions_
RESULTS
AND DISCUSSION
The electronic spectrum of methyl pyridinato cobaloxime in benzene solution was found to be temperature dependent (Fig. 1) as found previously for the cobalamin derivatives_ The temperature dependence of the electronic spectra of the cobaloximes could be the result of several factors. These include (a) a shift in the spectrum as a result of longer bond distance from increased vibrations and hence a lower ligand field at higher temperatures, (b) dimer formation with the elimination of the pyridine ligands, (c) ligand exchange. (d) equilibrium between hexacoordinated and pentacoordinated forms, (e) nonplanar cobalt, or(f) inner and outer pyridinato complexes_ The presence of isosbestic points in the electronic difference spectra of alkyl cobaloximes at various temperature differences in Fig. 1 indicates two separate spectral species and immediately rules out the first possibility because a continuously decreased ligand field with temperature would cause a continuous shift in the spectrumThe dimer of methyl cobaloxime is known (81 and is a possible explanation for the temperature-dependent spectrum in benzene_ Dimer formation, &and exchange (which must be impossible in benzene solution), and an equilibrium between hexacoordinate and pentacoordinate forms ah require the loss of a
pyridine molecule. To determine whether an equilibrium occurs in which the pyridine is lost, temperature-difference spectra were taken of methyl-pyridinato-cobaloxime in
ELECTRONIC SPECTRA OF ALKYL COBALOXIMES
101
FIG. 1. The temperature difference spectra of 2.5 X 10-3M methyl-pyridinato-cobalosime. The solvent was benzene which was deaented with nitrogen. The temperatures in each cell was 1” and Z’, lo and 7O, 1” and 12”, 1” and 15”, 1” and 20°, 1” and 29, 1” and 30° as we proceed from the smallest to the largest deviations_ The ordinate is a linear optical density scale and the abscissa is a linear waveIen$h scale.
benzene containing increasing pyridine concentrations from 1% to 90%. The experimenta data (Fig. 2) show no changes with pyridine concentration. These results ehminate the possibility of an equilibrium with either a pentacoordinated There are spectral differences between primary and secondor dimeric forms. ary alkyl cobalamins as a result of a change in position of the cobalt atom. [9] _ Cobalt(i)cobinamide can be alhylated by a bulky substituent only when a strong ligand in the sixth position is absent. Accordin g to Brodie this would allow the cobalt to remain above the pIane of the corrin system_ ConverseIy, the presence
A. BLANCO-LABRA, et ai.
III
WAVELENGfH (nm) I
I
I
I
I
I
I
I
I
I,
32@340360380400420440460480Mo520540660580600
I,,
FIG. 2. The temperaturedifference spectra of 0.0005 methyl-pyridinato-cobaioximein benzene-pyridinesolutions at temperaturesof 10°C vs 25°C. Dotted line is the difference spectrum in a solution containing 1% pyridine, the solid line in 40% py-ridine and the dashed line in 90% pyridine. The ordinateis a linearoptical density scaleand the abscissais a linear wavelengthscale.
of a strong ligand (e.g., 5,6-dimethylbenzimidazol or pyrimidine) would tend to force the cobaIt into the plane of the corrin, and this precludes the formation of a carbon-cobalt bond with a bulky allcyl group. Brodie [9] did not observe these two forms in equilibrium in the same compound but such an equilibrium between two positions for the cobalt atom would be another possible explanation for the temperature-dependent
the cobaloximes. In order to test this as a possible explanation spectrum, the Emax (445468
spectra of
for the temperature-dependent
nm) of the temperature-difference
spectra were
compared for the methyl, ethyl, and isopropyl derivatives expecting that if the two existing forms in the equilibrium correspond to the forms with the cobalt in the plane of the ring and out of the plane. then it would be expected that the proportion of the high-ener,~ form (out of the plane) would greatly increase in the series; methyl, ethyl, and isopropyl as steric hindrance between the alkyl group and the cobaloxime plane is increased_ However, no trend of this type was found (ef_, Fig- 3). These results indicate that the temperature dependent spectra is not the result of the existence of conformational isomers with the cobalt atom in and out of the plane. The only logical explanation remaining is that the temperature-difference spectrum for the alkyl-pyridinatocobaloximes in benzene solution is the result
ELECXRONIC SPECTRA OF ALKYL COBALOXIMES
103
difference spectra (2.5 x lo-” M benzene solution deaerated with nitrogen) methyl (max 390,455) c.45 = 323. isopropylpyridinato-cobaloxime (max 395,456) eq5 s = 28, temperature 30” and 10“. The ordinate is a linear optical density scale FIG. 3. The temperature
and the abscissa is a linear wavelength
scale.
of an equilibrium between an inner complex with a short cobalt-pyridine bond and an outer complex with a long cobalt-pyridine bond as suggested by Hill et al. [ 101 for the cobalamins. The original definitions of inner and outer complexes were in terms of orbital hybridization [ II] _ An inner complex was considered to be bonded by hybrid orbitals formed from d-orbitak with s- and p-orbitals of a higher she11 whereas an outer complex was considered to be bonded by hybrid d-,s-,and p-orbitals ail in
104
-4. BLANCO-LABRA,
et al.
the same shell. Other authors [I21 use the terms “ionic” ad “covalent” as synonomous with inner and outer complexes. The long cobalt-pyridine bond must certainly be less covalent and probably would utilize higher orbitals but could not in the usual sense be considered to be ionic although small charge separations wouId contribute to stability in benzene solution_ An exact description would require a molecular orbital calculation. Equilibrium between inner and outer complexes are known for other compounds and in particular for nickel complexes [ 131. The direction of the spectral shifts between inner and outer complexes is in agreement with our knowledge [ 141 of the electronic structure of the cobaloximes. it is well known that the primary effect of the axial ligand is to lower the energy of the highest occupied molecular orbital. Therefore, all transitions from the highest occupied moIecular orbital would be shifted to a lower energy when weakly interacting axial ligands are replaced by strongly interacting ones as is observed in the temperature-difference spectra. The difference spectra of methyl-aquo-cobaloxime and methyl-pyridinatocobaloxime (Fig_ 4) shows a marked similarity to the temperature-difference spectra of the methyl-pyridinato-cobaloxime. The difference between the aquo and pyridinato derivatives is probably similar to the difference between an inner and outer complex for pyridine because the aquo complex would be weakly held with little x-bonding as in an outer pyridinato complex. Of interest is the observation that the aquo complex also exists as an equilibrium between tier and outer complexes_ In Fig_ 5 are shown the temperature-difference spectra of methyl-aquo-cobaloxime in aqueous solution_ In order to make sure that the temperature-difference spectrum is not caused by conformational changes or ionizations in the hydrogen bridges between the dimethyl glyoxime ligands the cyclic oxime boronic ester of methyl-pyridinato-
FIG. I The concentration difference spectrum of I ml of a satrmated aqueous solution of methyl-pyridinato-cobaloxime each in a l-ml and a S-ml cuvette with the 5-ml cuvette diluted to capacity with water_
ELECTRONIC
SPECTRA
FIG. 5. The temperature
OF ALKYL
COBALOXIhlES
difference spectra of 2.5 X lo-’ M methyl-aquocobaloxime solvent was water deaerated with nitrogen. The temperature was IO” and 30°C. ordinate is a linear optical density scaIe and the abscissa is a linear wavelenth scale.
105
the The
cobaloxime was prepared by treatment with boron trifluoride Cl51 _ This compound also exhibited a temperature-difference spectra. The spectrum shown in Fig- 6 was identical for solutions of composition between 70% pyridine-30% benzene (v/v) to 100% pyridine. Below 70% pyridine the compound became too insoluble for good spectra to be obtained_ In order to estimate the amount of the outer sphere complex, the equilibrium between methyl-pyridinato-cobaloxime and methyl-methanolato-cobaloxime was measured in methanoI. Concentration difference spectra were measured by using 1 cm and 5 cm path length cuvettes with the same amount of methyl-pyridinato-cobaloxime in each cuvette but fiied to capacity with methanol_ Again the difference spectra
A. BLANCO-LABRA,
106
I1
360
IllI
400
1
I,‘,
440 480 WAVELENGTH
et ai.
I,
520
560
600
(nm)
FIG. 6. The temperature difference spectrum of 5 X 10m4Mboron fluoride etherate of methyl-pyridinato-cobaioximein 80% pyridine-20% benzene solution (v/v)_ The temperatureswere 9.8”C and 20°C. appears
very similar to the temperature-difference
spectra. The concentration
dependence of the peak at 445 nm in methanol is shown in Table 1. This data treated by the method of Viale and KalIen [ 161 gives an equilibrium constant of 2,740 t 5% for the reaction of methyl-methanolato-cobaloxime with pyridine to form methyI-pyridinato-cobaloxime_ The value of this equilibrium combined with a knowledge of the AOD between the two cells allowed us to calculate a value of the difference in extinction coefficients for the methanolato and pyridinato complexes of approximately 760. if we assume this same difference in extinction coefficient between the tier and outer complexes of pyridine and we neglect
the amount
this temperature
of outer complex at 1OC (the extinction coefficient at is assumed to be that of the inner complex), we can estimate
the amount of outer complex form shown in Table 2. The actual amount of outer form would be increased over these values by any outer form at i°C_ From the temperature dependence of the amount of outer form, the &!Z for the difference between outer and inner complexes can be estimated to be about 8.8 kml per mole. DISCUSSION
We do not intend to extrapolate these results to explain all the temperaturedependent
spectra observed for the cobalamins aithough the behavior of the
107
ELECTRONIC SPECTRA OF ALKYL COBALOXIMES TABLE
1
The Absorption Differences at 445 nm of I ml of Methyl-Pyridinato-Cobaloxime in Methanol at the Concentration Listed in Both a l-ml and a S-ml Cuvette with the 5-ml Cuvette Diluted to Capacity with Methanol_ Concentration
of
(X 104)
A Absorbance 0.360 0.123 0.048 0.032 0.0195 0.0085
14.5 5.83 2.91 2.33 1.75 1.16
TABLE 2 The Equilibrium Constant for the Formation of the Outer Complex Form of Methyl-Pyridinato-cobaIoxime and the Percent of Material in the Outer Complex Form at Various Temperatures in Benzene Solution.
AOD
T°K (vs T1 = l°C)
(445 nm X 104)
275 280 285 288 293 298 303
46 73 88 112 154 185 242
KeqX IO3
c/oOuter form
2.4 3.6 4.7 5.9 8.2 9.8 12.9
0.24 O-38 0.46 0.60 0.81 0.97 1.27
temperature-difference spectra and the similarity of the high-temperature form to the aquo derivative [ l] is analogous to the results reported here. There are probably several explanations depending on the conditions for the temperaturedependent spectra of the cobalamins. However, alkyl-pyridinato-cobaloximes in benzene solution appear to exist in a temperature-dependent equiiibrium between inner and outer forms. The outer form is in low concentration relative to the inner complex form. Exchanges of the fifth ligand are likely to be very important for many functions of vitamin B 1Z [ 171. An example is the proposed mechanism for methane production [ 181, which first requires exchange with a mercaptan before cleavage of the carbon-cobalt bond. Exchange reactions at the 5th ligand position in methyl-aquo-cnbaloxime is 25.6 kcal; a much larger value than our estimate for the formation of an outer complex. Therefore, we visualize
108
A. BLANC&LABRA.
et al.
a rapid reversible equihbrium between the inner and outer complexes followed by a rate-determining step for dissociation of the outer complex_ Because the outer complex is a considerably higher energy than the inner complex, the outer complex wilI dissociate faster than the inner complex_ If the enzyme were capable
of holding
the fifth
ligand
in an outer
complex,
the exchange
reactions
would be very much more rapid. If true, this would be another example of an entatic state of a metallo enzyme [20] in which the strain produced by the
protein increased the reactivity of the active penter. We note that spectral changes We 08285
are observed
wish
to
when cobalamins are bound to an enzyme
acknowledge
and a fellowship
from
the
aid
of
COivACYT
U.S. {from
Public Mexico)
Health
[ 10) _ Service
Grant
GM
for A-B-L_
REFERENCES I_ J_ Fo_x, R_ Banninger, R. Draper and L. L_ ingraham, Arch_ Biochem Biophys- 125, 1022 (1965)_ 2. R_ A_ Firth, H- A. 0. Hill, B. E_ Maan. J_ $1. Pratt, R_ G. Thorp, and R. J. P_ Williams,J.
them_ Sot_ (A), 2419 (1968). 3_ T_ Darwin,J. Am. Chem. Sot. 93,2629 (1971). 4. H. A. 0. Hill, B. E. Mann, J. M. Pratt and R. J. P. Williams, J. Chem. Sot. (A), 564 (1968). 5. R. A. Firth, H. A. 0. Hill, J. M. Pratt, R. J. P. Williams and W. R. Jackson, Biochemistry, 6,2178 (1967)_ 6_ G. N_ Scfuauzer, Act. Chem_ Res. 1,97 (1968). 7. G. N. Schrauzer, L. l? Lee and J. iti Sibert, Act. Chem. Res. 92.2997 (1970). 8. G. N. Schrauzer and R. J_ Windgassen,J_ Am Chem. Sot_ 88,3738 (1966). 9. J. D. Brodie. &cc_ Nat Acad. Sci. 62,461 (1969). lo_ H_ A_ 0. Hill. J_ M. Pratt and R_ J. P_ Williams, Chem Brit. 156 (1969). 1I. H. Taube, them. Rev_ 50,69 (19.52). 12_ F_ H_ Burstailand R. S. Nyho1m.J. Chem. Sot, 3570 (1952). 1K H. Brintzinger and G. C. Hammes, Inorg. Uzem. 5,1286 (1966). 14. G. N. Schrauzer, Natwwfisenshaften 53,459 (1966). 15. G. N. S&rawer and R. J. Windgassen,J. Am. Chem. Sot_ 88,3738 (1966). 16. 17_ 18. 19. 20_
R. 0. Viile and R_ G. Kallen, Arch_ Biochem. Biobhys. 146.271 (1971) G_ Schnauzer et al_. Bioinorg. Chem 2 (2), 93 (1973). J. W_ Sibert and G. N. Schrauzer,J. Am Chem Sot. 92,142l (1971). K. Brown and R. G. Alien, J. Am Chem Sot. 94, 1894 (1972). B. L_ VaJlee and 2_ J_ P_ WilJiams,~oc_ Nat AC&_ Sci 59,498 (1968).
Received lriay 24. I 9 74; revised July I, I 9 74
