(Received for publication, September 3, 1996)
From the Department of Biochemistry, University of
California, Riverside, California 92521-0129, the § Novo
Research Institute, Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark, and
the ¶ Department of Chemistry, University of Copenhagen,
DK2100 Copenhagen, Denmark
Magnetic circular dichroism (MCD) spectra of a
series of adducts formed by the Co(II)-substituted R-state insulin
hexamer are reported. The His-B10 residues in this hexamer form tris
imidazole chelates in which pseudotetrahedral Co(II) centers are
completed by an exogenous fourth ligand. This study investigates how
the MCD signatures of the Co(II) center in this unit are influenced by
the chemical and steric characteristics of the fourth ligand. The
spectra obtained for the adducts formed with halides, pseudohalides, trichloroacetate, nitrate, imidazole, and 1-methylimidazole appear to
be representative of near tetrahedral Co(II) geometries. With bulkier
aromatic ligands, more structured spectra indicative of highly
distorted Co(II) geometries are obtained. The MCD spectrum of the
phenolate adduct is very similar to those of Co(II)-carbonic anhydrase
(alkaline form) and Co(II)--lactamase. The MCD spectrum of the
Co(II)-R6-CN
adduct is very similar to the
CN
adduct of Co(II)-carbonic anhydrase. The close
similarity of the Co(II)-R6-pentafluorophenolate and
Co(II)-R6-phenolate spectra demonstrates that the
Co(II)-carbonic anhydrase-like spectral profile is preserved despite a
substantial perturbation in the electron withdrawing nature of the
coordinated phenolate oxygen atom. We conclude that this type of
spectrum must arise from a specific Co(II) coordination geometry common
to each of the Co(II) sites in the Co(II)-R6-phenolate,
Co(II)-R6-pentafluorophenolate, Co(II)-
-lactamase, and
the alkaline Co(II)-carbonic anhydrase species. These spectroscopic
results are consistent with a trigonally distorted tetrahedral Co(II)
geometry (C3v), an interpretation supported by the
pseudotetrahedral Zn(II)(His)3(phenolate) center identified
in a Zn(II)-R6 crystal structure (Smith, G. D., and Dodson,
G. G. (1992) Biopolymers 32, 441-445).
Several structural features frequently characterize the catalytic
sites of zinc enzymes. These features comprise a distorted tetrahedral
geometry incorporating one or more histidine groups and a coordinated
"activated" water molecule oriented out into a cavity or cleft
which forms the substrate binding site on the enzyme surface (1, 2).
The R-state1 insulin hexamer represents a
well characterized biomolecular analog of this type of coordination
unit (3, 4). The metal chelate site in the R-state insulin hexamer
comprises a pseudotetrahedral array of three histidines and one
exogenous small molecule ligand as shown in Fig. 1. The tris
imidazole-Zn(II) motif occurs at the active site of several
structurally characterized zinc metalloenzymes, including the carbonic
anhydrases (Zn(His)3(H2O)), -lactamase (Zn(His)3S-Cys), astacin
(Zn(His)3(Tyr)(H2O)),
DD-carboxypeptidase (Zn(His)3(H2O)), and the metzincins (5, 6, 7, 8, 9). The
analysis of spectroscopically congruent transition metal-substituted metalloprotein derivatives has provided revealing insights into the
active site structure and mechanistic details of many functionally diverse metalloproteins (1, 10, 11, 12). Despite the importance of such
derivatives and their small molecule synthetic analogs, to our
knowledge, there are no well characterized examples of small molecule
complexes that stabilize pseudotetrahedral tris imidazole coordination
with Zn(II) as well as with the chromophoric metal ion probes Co(II)
and Cu(II).
The R-state insulin hexamer is one system that has been shown to stabilize pseudotetrahedral M(His)3L (M = Zn(II), Co(II), Cu(II)) coordination in which the fourth ligand site may be occupied by a wide range of exogenous small molecule ligands (L) (3). This hexamer thus represents a unique and highly pertinent spectrochemical model system for the investigation of the structural and spectroscopic interrelationships of this type of metal center. A major difficulty with biological chromophores is the fact that metalloproteins usually impose low symmetry ligation at the active sites which result in Co(II) derivatives that are difficult to model both theoretically and with small molecule Co(II) complexes. Furthermore, the high spin Co(II) ion in a low symmetry environment gives rise to a particularly complex splitting of the ligand field bands due to the low symmetry geometry in concert with several different electronic effects (13). The difficulty in accounting for the ligand field components observed experimentally has highlighted the importance of Co(II) model systems that incorporate biologically relevant ligands into nonclassical chelate geometries that may be perturbed in a systematic manner.
The insulin hexamer consists of three dimeric units associated about a
3-fold symmetry axis thus forming a torus-shaped molecule (14). The
hexamer has been shown to exhibit allosteric ligand binding
characteristics that involve two classes of sites as follows: (i) the
metal ion at the His-B10 site to which anionic ligands coordinate and
(ii) six hydrophobic pockets formed between subunits to which neutral
phenolics bind (Fig. 1) (3, 15, 16, 17, 18). Three conformationally distinct
allosteric forms of the zinc insulin hexamer have been characterized
crystallographically (4, 19, 20, 21). These forms have been designated as T6, T3R3, and R6 where
the T-state refers to a subunit conformation in which the B-chain
residues B1-B9 exist in an extended conformation, whereas in the
R-state these residues adopt an -helical conformation (15). The
hexamers bind Zn(II) ions at two sites; each site is formed by a set of
three symmetry-related His-B10 residues. In the T6
structure (19) and in the T3 half of the
T3R3 structure (20), the T-state subunits form
an octahedral Zn(II) site comprising the three His-B10 nitrogens and
three water molecules. In the R6 structure (4, 21) and in
the R3 half of the T3R3 structure, the three His-B10 nitrogens form a pseudotetrahedral (C3v)
Zn(II) site completed by a fourth ligand from solution
(Fig. 1A). The three symmetry equivalent
B1-B10
-helices form a cylindrical channel to the metal ion along
the central 3-fold axis of the R6 hexamer.
Ligand binding studies of Co(II)-insulin hexamers in solution have established that saturating concentrations of certain phenolic compounds, in conjunction with the heterotropic effects of anionic ligands that bind to the Co(II) sites, displace the equilibrium strongly in favor of the R6 conformation (16, 22). As part of our continuing studies to characterize the spectrochemical properties of transition metal-substituted insulin hexamers and the structures of the R-state species, we have measured the MCD induced in the visible absorption bands of a series of Co(II)-R6 hexamer complexes.
The chemicals used in these studies were reagent grade or better and were used as supplied. Metal-free human insulin was supplied by the Novo Research Institute (Denmark).
InstrumentationMCD spectra were measured with an
instrument based on a Cary 14 double monochromator, a
MgF2 polarizer (Karl Lambrecht), a CaF2
modulator (Hinds International), and a lock-in amplifier (SR 510, Stanford Research Systems) under the control of a PC-AT microcomputer.
This instrumentation was used in conjunction with a superconducting
magnet (Spectromag III, Oxford Instruments) producing a magnetic flux
density of 4 T (1 T = 10,000 G) or an electromagnet producing a
magnetic flux density of 1.2 T. Quartz cells with path lengths of 0.1 and 0.8 cm were used with the 1.2- and 4-T magnets, respectively. The
MCD spectra have been corrected for natural optical activity.
Electronic absorption spectra were recorded on a Perkin-Elmer -17
spectrophotometer. All measurements were conducted at room temperature.
MCD is reported as the molar magnetic ellipticity,
[
]M, and is expressed in units of degrees ·cm2 dmol
1 G
1. The SI unit is m2 mol
1 T
1; 1 deg·cm dmol
1
G
1 = 4
m2(18ln10)
1 mol
1
T
1.
The preparation and electronic absorption spectra of a wide variety of the anion complexes formed by the Co(II)-R6 hexamer have been reported previously (22). Spectra were recorded in 50 mM Tris-ClO4 buffer in the presence of 100 mM phenol at pH 8.0, except for complexes of the weakly binding anions, nitrate, acetate, and trichloroacetate which were recorded at pH 7.5 and phenolate itself, which was recorded at pH 8.9. These conditions were chosen to drive, as completely as possible, the competing equilibria toward formation of the Co(II)-R6 complex of interest. Under these conditions the visible absorption spectrum is dominated by a single, pseudotetrahedral Co(II) chromophore corresponding to a Co(II)-R6-anion complex (16, 22). Electronic absorption spectra were recorded on each sample immediately prior to and following the MCD measurements to monitor sample stability. No significant differences were observed, except in the cases of imidazole and 1-methylimidazole where some diminution of the absorbance spectra occurred. For this reason the extinctions and MCD intensities for these complexes are reported as approximate values.
Previous work (22) has shown that the spectral profiles of
the electronic transitions in the visible region are very sensitive to
the nature of the small molecule ligand coordinated to the Co(II) ion
of R6 adducts. The MCD and electronic absorption spectra of
the Co(II)-R6-phenolate complex are presented in
Fig. 2, A and B. The absorption
spectrum exhibits four bands located respectively at 508, 543, 622, and
641(sh) nm. The MCD induced by a 4 T magnetic field resolves these
features as a positive band at 498 nm accompanied by negative bands at
538, 613, and 629(sh) nm. An additional small positive band occurs at
465 nm. In view of the special significance of this spectrum as a
spectroscopic model for the hydroxide ion adduct of Co(II)-substituted
carbonic anhydrase (Co(II)-CA), the 4 T magnet was used in an attempt
to identify possible fine structure in the MCD bands. However, the MCD
induced by the 4 T magnetic induction did not resolve any additional
spectral features when compared with the 1.2 T experiment. The
remaining MCD spectra reported herein were recorded with a 1.2 T
magnetic flux density. The spectra of the
Co(II)-R6-pentafluorophenolate complex (Fig. 2,
C and D) display profiles analogous to those of
the Co(II)-R6-phenolate complex. In each case, the
absorption spectra comprise envelopes split into two high energy bands
and two lower energy bands. In the electronic absorption spectrum of
the Co(II)-R6-pentafluorophenolate complex, bands occur at
524(sh), 543, 606 and 629(sh) nm. These are distinguishable in the MCD
spectrum as a positive band at 503 nm and negative bands at 538, 595 nm, and 621(sh) nm with an additional small positive band at 465 nm.
The structure and steric bulk of the phenol and pentafluorophenol
molecules are very similar; therefore, it can be reasoned that the
differences in the spectra of Fig. 2, A and B and
C and D, originate from the different donor
properties of the respective coordinated O atoms.
Halides and Pseudohalides
The absorption spectrum of the
Co(II)-R6-CN adduct (Fig. 3,
A and B) is clearly quite different from those of
the phenolate and pentafluorophenolate adducts. It is narrower, more
intense, and displays only two absorption features. The MCD spectrum
resolves these features into a positive band at 529 nm and a negative
band at 575 nm. The qualitative appearance of this spectrum exemplifies many of the adducts reported in Table I for which the
actual spectra are not shown (viz. imidazole,
1-methylimidazole, chloride, bromide, trichloroacetate, nitrate, and
azide). Specifically, these spectra comprise a single positive band
accompanied by a single negative band at lower energy. The spectra of
Fig. 3, A and B, are almost identical to the
respective absorption and MCD spectra of the
Co(II)-CA-CN
adduct (23).
|
The spectra of the Co(II)-R6-OCN adduct are
presented in Fig. 3, C and D. The electronic
absorption spectrum comprises three distinct components. The MCD
spectrum displays two prominent features, a negative band at 585 nm and
a positive band at 524 nm. A poorly resolved shoulder on the negative
band is also evident at approximately 556 nm. The MCD spectrum of the
iodide adduct is very similar to that of the cyanate adduct shown in
Fig. 3, C and D.
The acetate, trichloroacetate, sorbate, and nitrate ligands form a category that may be considered separately. These anions are nonlinear ambidentate ligands that possess multiple oxygen donor atoms. The spectra of the Co(II)-R6-acetate adduct are presented in Fig. 3, E and F. The distinctive profile of the acetate spectrum suggests that the Co(II) coordination in this adduct is somehow different from the other complexes surveyed in this study. One attractive explanation for this is the possibility that the acetate group acts as a bidentate ligand producing a five-coordinate Co(II) center or a four-coordinate center with an additional weaker Co(II)-O interaction. An example of this latter coordination has been characterized crystallographically in the small molecule complex bisacetato(ethylenethiourea)-cobalt(II) (24). The Co(II) geometry in this complex is distorted tetrahedral with two additional long Co(II)-O bonds. The electronic spectrum of this complex could be rationalized in terms of a pseudotetrahedral Co(II) center, but the presence of the weak Co-O bonds were shown to have a significant effect on the electronic structure of the Co(II) ion. The sorbate and trichloroacetate ligands afford an interesting comparison to acetate because these ligands possess the same donor functionality. The intensities and spectral energies for both the absorption and MCD spectra are very similar for the sorbate and acetate complexes, whereas the trichloroacetate spectra are clearly distinct and more comparable with those of the halide and pseudohalide complexes. The trichloroacetate and nitrate spectra (data not shown) probably represent near tetrahedral Co(II) centers.
BenzenethiolateThe spectra of the Co(II)-R6-benzenethiolate adduct are presented in Fig. 3, G and H. The visible absorption spectrum comprises two well separated bands that are resolved into an intense negative MCD band at 617 nm with a shoulder at 575 nm and a positive band at 526 nm. The higher energy LMCT bands are resolved into a positive MCD band at 427 nm and a negative band at 377 nm.
Geometrical Deviations and Transition EnergiesTable I lists
the approximate energy of the 4T1(P)
4 4A2 transition for each of
the adducts as estimated from the mid-point between the major positive
and negative visible MCD bands. For the Co(II)-R6 spectra
that correspond to near tetrahedral geometries, the trend in this
transition energy is I
< Br
< Cl
, N3
< NCO
, CN
, 1-methylimidazole < imidazole. The halides and pseudohalides in this group conform to the
order predicted by the spectrochemical series. The larger transition
energies of the imidazole and methylimidazole adducts indicate that the
bonding interaction between the Co(II) ion and the exogenous imidazole
group must involve considerable covalency.
MCD spectroscopy is based on the Faraday effect which gives rise
to the appearance of optical activity in a sample that has been exposed
to a longitudinal magnetic field (25). The MCD arises as a consequence
of the interaction of the electronic energy levels with the applied
magnetic field, whereas the CD arises as a consequence of the
structural asymmetry of the chromophore itself. Studies of structurally
characterized small molecule Co(II) complexes have established that the
spectral properties of the high spin Co(II) ion are very sensitive to
the ligand field strength and the geometrical disposition of the donor
atoms. The intensity, energy, and degree of structure associated with
the visible absorption envelope can provide a useful indication of the
stereochemistry (25, 26). This property makes the Co(II) ion a useful
optical probe of active site structure in metalloproteins for which
Co(II)-substituted derivatives can be prepared. Octahedral Co(II)
complexes possess weakly intense bands at approximately 500 nm ( < 100 M
1 cm
1), whereas the
visible absorption bands of tetrahedral complexes occur at low energy
and are considerably more intense (
> 300 M
1 cm
1). The spectral features
of five-coordinate Co(II) centers are less diagnostic but are generally
observed to be less intense than those of tetrahedral complexes
(150 <
< 300 M
1
cm
1). Three ligand field transitions are expected for
Co(II) ions possessing tetrahedral symmetry. The lowest energy
transition, 4T2(F)
4A2, occurs in the 3000-5000 cm
1
region and is usually not observed. The second transition,
4T1(F)
4A2, occurs
in the near-infrared region, and the third transition, 4T1(P)
4A2, occurs
in the visible region, 715-500 nm. Our MCD experiments were restricted
to the visible spectral region incorporating the 4T1(P)
4A2
transition. In distorted tetrahedral environments, the low symmetry
splitting of the 4T1(P) level results in a
splitting of the 4T1(P)
4A2 absorption envelope that provides a
qualitative indication of the deviation from tetrahedral symmetry.
Detailed interpretations of Co(II) electronic spectra are complicated
by the effects of the low symmetry ligand field in addition to
spin-orbit coupling, Jahn-Teller effects, and the possibility of spin
forbidden transitions. Definitive correlations of the electronic and
geometric structures of Co(II) chromophores require single crystal
spectral measurements to be made directly on crystallographically
defined Co(II) sites. For metalloproteins, such studies are
inconveniently labor intensive and are generally technically
infeasible. Consequently, much emphasis has been placed on making
qualitative comparisons with small molecule Co(II) complexes,
particularly those containing biologically relevant ligands such as
thiolates, carboxylates, and imidazoles. The value of the
Co(II)-R6 hexamer as a spectroscopic model arises because (i) pseudotetrahedral Co(II)His3L systems are rare, (ii)
the fourth exogenous ligand, L, may be varied extensively, and (iii)
several crystal structures have been reported defining the
pseudotetrahedral Zn(II)His3L sites of
Zn(II)-R6 or Zn(II)-T3R3 hexamer
complexes (4, 20, 21, 27, 28, 29).
The MCD spectra recorded for the
Co(II)-R6 complexes formed with halides and pseudohalides
exhibit two major bands, one positive at higher energy and the other
negative at lower energy. This type of spectrum is exemplified by that
of the Co(II)-R6-CN adduct (Fig. 3,
A and B) that corresponds to a Co(II) geometry close to regular tetrahedral symmetry. This conclusion is supported by
the MCD spectra observed for structurally characterized tetrahedral Co(II) complexes, such as Co(py)2Br2 (where py
is pyridine), [CoCl4]2-, and
[Co(SCN)4]2- (30). In contrast, the
Co(II)-R6 complexes formed with phenolate, pentafluorophenolate, and benzenethiolate possess spectra that exhibit
three or four clearly resolved major bands, as shown in Fig. 2,
A-D, and Fig. 3, G and H,
respectively. These spectra may arise from Co(II) centers that possess
coordination geometries appreciably distorted from tetrahedral. Several
structurally characterized small molecule complexes with distorted
tetrahedral Co(II) centers that exhibit somewhat comparable electronic
spectra have been reported (31, 32).
The His-B10 metal chelate site in the R-state insulin
hexamer bears some structural similarity to the active sites in several zinc enzymes. We have reported previously that the absorption spectra
for many of the Co(II)-R6 complexes are remarkably similar to those of the corresponding adducts formed with Co(II)-carbonic anhydrase (33). The metal chelate site in CA consists of three histidine residues and is completed by one or two small molecule ligands (5). The spectroscopic and crystallographic data for Zn(II)-
and Co(II)-CA indicate that the metal center may accommodate expanded
coordination numbers; thus pseudotetrahedral and pentacoordinate geometries are possible for Co(II)-CA (26, 34). In the absence of added
anions at alkaline pH values, the absorption and MCD spectra of
Co(II)-CA exhibit maxima at 518 nm (+), 556 nm (), 617 nm (
), and
641 nm (
). Although the interpretation of Co(II)-CA spectra has been
complicated by uncertainty regarding equilibria involving multiple
forms, the exogenous coordinated anion in this species has been
postulated to be a hydroxide ion. The distinctive appearance of the
Co(II)-CA spectrum has fueled ongoing speculation that it signifies the
mechanistically relevant structural motif of a key intermediate in the
HCO3
dehydration pathway for Co(II)-CA
(23, 35). The large splitting between the bands of the alkaline
Co(II)-CA spectra has been interpreted as indicative of a trigonally
distorted tetrahedral Co(II) geometry (23). This interpretation is
supported by comparison of the MCD spectra of a series of
five-coordinate Co(II)-CA adducts with that of alkaline Co(II)-CA (36).
The highly similar electronic and MCD spectra observed for the
Co(II)N(His)3O(phenolate),
Co(II)N(His)3O(hydroxide), and
Co(II)N(His)3S(Cys) centers of the Co(II)-R6
hexamer, alkaline Co(II)-CA, and Co(II)-
-lactamase, respectively,
suggest that the distinctive nature of these spectra originate
primarily from the effect of a specific Co(II) geometry. For the
Co(II)-R6-phenolate complex, the effect of increasing the
electronegativity of the coordinated phenolate oxygen atom is
demonstrated by examining the spectra of the
Co(II)-R6-pentafluorophenolate adduct (Fig. 2, C
and D). The more electron withdrawing pentafluorophenolate oxygen results in electronic and MCD spectra that possess profiles substantially analogous to those of the Co(II)-R6-phenolate
spectra. The consequence of this large perturbation in the donor
properties of the phenolate oxygen atom is manifested as relatively
minimal changes in the splitting of the MCD spectral components and the transition energy (Table I). The observation that the Co(II)-CA-like electronic and MCD spectral profiles are preserved, in spite of this
perturbation, confirms that these distinctive spectral features are
related intimately to a specific type of Co(II) coordination geometry.
The two low energy bands have been assigned to transitions to the
doublet states 2T1(G) and 2E(G)
which can become enhanced by spin orbit coupling only. Via spin-orbit
coupling, a trigonal distortion of the Co(II) geometry is expected to
give rise to an increased mixing of these doublet states into the
quartet states. It is noteworthy that the symmetry of the M(II) chelate
site in the M(II)-R6 hexamer is predisposed toward a
trigonally distorted tetrahedral geometry because the cylindrical
channel to the metal is expected to constrain a bulky fourth ligand,
such as the phenolate group, to an orientation along the 3-fold
symmetry axis of the hexamer. Such an arrangement is observed for the
Zn(II)(His)3-phenolate center in the monoclinic Zn(II)-R6 crystal structure (21).
The interaction of phenol with Zn(II)-CA and Co(II)-CA has been studied
in detail because phenol is known to be a competitive inhibitor of the
CO2 hydration reaction catalyzed by CA. Phenol molecules
have been shown to bind to CA; however, the phenolate anion does not
displace the metal-bound hydroxide ligand of either Co(II)-CA or
Zn(II)-CA, even at high pH values (37, 38). The phenolate-binding
affinities of the metal centers are clearly very different for the
M(II)-CA and M(II)-R6 species even though our results show
that the structures of the respective Co(II) centers must be extremely
similar for at least some of the adducts, notably CN.
This observation suggests that the residues forming the outer coordination sphere must play a critical role in defining the coordinative selectivity of the active site metal ion in CA.
In -lactamase, three histidines and a
cysteine have been identified as ligands to the active site
Zn2+ (8). The Co(II) derivative of
-lactamase exhibits
absorption and MCD spectra that are remarkably similar to those of
alkaline Co(II)-CA. However, there is some conflict regarding the
interpretation of these spectral features as indicative of either a
pentacoordinate Co(II)His3(S-Cys)(H2O)
arrangement (11) or a distorted tetrahedral Co(II)His3(S-Cys) arrangement (39). The putative
coordinated water of the former arrangement has been postulated to play
a key role in the mechanistic pathway of
-lactamase catalysis (11). In view of the strong spectral congruency between
Co(II)-R6, Co(II)-CA and
-lactamase, it is reasonable to
infer that the spectra observed for Co(II)-
-lactamase are consistent
with a distorted tetrahedral (C3v)
Co(II)His3(S-Cys) donor set. Although an activated water molecule may well play a role in catalysis, this spectral
interpretation suggests that in the resting enzyme the putative water
molecule does not interact with the Co(II) strongly enough to
significantly influence the electronic structure of the Co(II) ion.
Co(II)-substituted blue
copper proteins possess visible absorption spectra indicative of highly
distorted tetrahedral Co(II) sites (40, 41). These spectra are in
accord with the x-ray crystal structures of blue copper proteins which
all identify low symmetry Cu(II) geometries comprising two histidines,
a methionine, and a cysteine as ligands. The native blue copper
proteins are characterized by small ESR hyperfine coupling constants
and intense visible S(Cys) Cu(II) charge transfer bands, features
uncommon for Cu(II) chromophores (42). The Cu(II)-R6
hexamer exhibits similar spectral features when a thiolate ligand
occupies the fourth coordination site (43, 44). Furthermore, the
visible absorption spectra of Co(II)-R6 thiolate complexes
are qualitatively very similar to those of Co(II)-substituted blue
copper proteins. Because the spectral properties of blue copper
proteins and the corresponding Co(II)-substituted derivatives have long
been considered highly distinctive signatures of the active site
stereochemistries and electronic structures, the spectroscopic analogy
with the M(II)-insulin hexamer is indeed remarkable. The MCD spectrum
of the Co(II)-R6-benzenethiolate complex (Fig.
3H) extends this analogy. The MCD spectrum of the
Co(II)-R6-benzene-thiolate complex appears to be very
similar to those of Co(II)-substituted azurin, -plastocyanin, and
-stellacyanin. The two transitions occurring in the UV region of the
Co(II)-substituted blue copper proteins have been ascribed to LMCT
transitions from the 3p
and 3p
orbitals of
the cysteine thiolate sulfur to the Cu(II) d vacancy (40,
41). The seemingly analogous transitions in the MCD spectrum of the
Co(II)-R6-benzenethiolate complex are resolved at 427 and
377 nm, a separation of 3100 cm
1. The splitting of the
and
orbitals will depend on the symmetry of the ligand field
and the donor properties of the ligating atoms. The value of 3100 cm
1 may be compared with the values of 3600-4900
cm
1 reported for the aforementioned Co(II)-substituted
blue copper proteins (41).
The Co(II)-substitution experiment for insulin (45, 46) has become widely utilized as a convenient method for spectroscopically characterizing the conformational behavior and metal-binding characteristics of insulin and its derivatives in solution. Co(II)-insulin crystals are isomorphous to Zn(II)-insulin crystals,2 and consequently, the Co(II)-hexamer appears to represent a good chromophoric facsimile of its biologically relevant Zn(II) counterpart. Several crystal structures have shown that, in Zn(II) hexamers, R3 trimers impose tetrahedral coordination upon the Zn(II) atom, whereas T3 trimers are observed to coordinate Zn(II) in an octahedral arrangement (4, 19, 20, 21). These structures show that the coordination geometry of the metal ion appears to correlate with the conformation of the subunits within each trimer. The identity and oxidation state of the metal ion at the His-B10 site have been shown to influence the conformational properties of the hexamer (15, 44, 46). These effects can be attributed to the unique coordinative preferences of the different metal ions. The d10 electronic configuration of the Zn(II) ion means that no ligand field stabilization energy is derived upon Zn(II) complexation. Consequently, the resulting Zn(II) stereochemistry is dictated only by the bonding and steric requirements of the ligating environment. A recent Zn(II)-insulin crystal structure (29) presents evidence for dual coordination about one Zn(II) atom in a Zn(II)-T3R3 type structure. In this structure, the Zn(II) ion of the T3 trimer exhibits both tetrahedral and octahedral coordination as a result of a 2-fold disorder. On the basis of this observation it was suggested that a similar situation could exist for Co(II) hexamers, thus calling into question the interpretation of spectroscopic results. However, it must be noted that, in contrast to Zn(II), the ligand field stabilization effect is expected to be a significant factor in determining the stereochemistry of the high spin Co(II) ion. Our MCD studies show that the orbital splitting of the Co(II) center in the Co(II)-R3 trimer is, indeed, significantly affected by the nature of the fourth ligand. The ligand field stabilization effect is expected to result in a degree of thermodynamic selection for Co(II) stereochemistry within the Co(II)-hexamer, and this selection will be modulated by the nature of the fourth exogenous ligand. The stabilization of the Co(II)-R6 hexamer via heterotropic ligand binding interactions involving the Co(II) ion has been reported (3, 16, 17, 18). We consider it unlikely, therefore, that a given insulin trimer conformation would simultaneously stabilize octahedral and tetrahedral Co(II) geometries in solution.
In conclusion, the His-B10 site in the M(II)-R6 hexamer
provides a fortuitous example of a biological chelate that stabilizes pseudotetrahedral transition metal coordination. This system has enabled us to examine a series of pseudotetrahedral
Co(II)(His)3L centers in which the variation of the
structural and electronic features of the Co(II) ion originates from
the variation in the nature of one exogenous ligand. Several factors
are expected to influence the geometry of the Co(II) chromophore in the
Co(II)-R6 hexamer, the steric bulk of the exogenous fourth
ligand, the electronic structure of this ligand, and its disposition of
donor atoms. The data of Table I show that near-tetrahedral geometries
are obtained with the halides, pseudohalides, trichloroacetate,
nitrate, imidazole, and 1-methylimidazole. Considerably distorted
geometries are obtained with the larger aromatic ligands. In order to
accommodate the additional steric bulk of these ligands, an alternative
arrangement of the B1-B10 residues is required which evidently results
in a distorted Co(II) geometry. For the near-tetrahedral adducts, the
transition energies are influenced by the different donor properties of
the ligands in conformity with the spectrochemical series. Although the
electronic absorption spectra of these adducts show considerable
diversity, the qualitative appearance of the MCD spectral profiles fall
into distinct categories. These ligand perturbation experiments
demonstrate that the MCD dispersion is highly sensitive to the Co(II)
coordination geometry and appears to be less affected by the actual
chemical nature of the exogenous ligand donor atoms in the
Co(II)(His)3L unit. The present study provides evidence for
a structural analogy between the Co(II) sites of
Co(II)-R6-phenolate, alkaline Co(II)-CA, and
Co(II)--lactamase.
Although structural conclusions based on spectral methods alone are
necessarily tentative, the spectra reported herein fall into distinct
spectral classes that almost certainly correspond to discrete Co(II)
coordination geometries. The intriguing spectral parallels with blue
copper proteins (3, 43, 44), carbonic anhydrase (33), and
Co(II)--lactamase attest to the utility of an artificial
metalloprotein system to model the unique spectrochemical and
structural features of spectroscopically unusual
metalloproteins.
We thank Karen Jørgensen for experimental assistance and Dr. Ole Hvilsted for generating the molecular graphics image of insulin.