Ground-State Properties of Heme Complexes in Model Compounds

Chapter 24

Ground-State Properties of Heme Complexes in Model Compounds and Intact Proteins Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on July 11, 2018 at 10:09:31 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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Frank U. Axe , Lek Chantranupong , Ahmad Waleh , Jack Collins , and Gilda H. Loew 2

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The Rockefeller University, 701 Welch Road, Suite 213, Palo Alto, CA 94304 SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025 The ground and low-lying spin states of f e r r i c porphy r i n (heme) complexes found in model compounds and intact proteins have been studied using an INDO-RHF-SCF method parameterized to include t r a n s i t i o n metals. The results for the model compounds using known c r y s t a l structure geometries are consistent with and help explain the o r i g i n of their observed electromagnetic properties. These studies demonstrate the a b i l i t y of this method to determine with a high degree of reliability the r e l a t i v e energies of the manifold of spin states of f e r r i c heme complexes. The same method has been used to address unresolved questions regarding the resting states of four heme proteins, cytochrome P450 which is a monofunctional oxidase, cytochrome c peroxidase (CCP) and catalase (CAT) both with peroxi dase oxidizing a c t i v i t y , and metmyoglobin (MMB) which lacks s i g n i f i c a n t peroxidase oxidizing a c t i v i t y . The characterization of the P450 resting state leads to a possible explanation of the o r i g i n of i t s low-spin (S = 1/2) form. This result helps resolve the apparent contradiction between the presence of water as an a x i a l ligand as determined by the x-ray structure and the absence of hyperfine s p l i t t i n g s in ESR spectra with 65% O enriched water. Comparisons of the resting state calculations of CCP and MMB provides an understanding of the origins of the differences in observed e l e c t r o magnetic properties for MMB and CCP in spite of the s i m i l a r i t y of their active s i t e s . Differences in function between MMB and CCP could not, however, be understood from properties of the active s i t e alone. Changing the imidazole ligand to an imidazolate in CCP makes i t s active s i t e more similar to CAT. Thus, the anionic form of the imidazole ligand of CCP, thought to be p a r t i a l l y formed by Η-bonding to a nearby Asp residue, could account at least in part for the similar a c t i v i t i e s of CCP and CAT as oxidizing enzymes. cam

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0097-6156/89/0394-0339$06.00/0 ο 1989 American Chemical Society Salahub and Zerner; The Challenge of d and f Electrons ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

340

THE CHALLENGE OF d AND f ELECTRONS

Heme proteins a l l share a common active s i t e or prosthetic group consisting of an iron-porphyrin (heme) unit, a nearly planar e n t i t y embedded i n the globular protein and connected to i t by one or at most two nearby amino acids which serve as a x i a l ligands. This important family of proteins performs three basic b i o l o g i c a l functions (1-5): reversible oxygen transport (globins), electron transfer (cytochromes), and metabolic oxidation of small organic molecules and peroxides (peroxidases, catalases, and cytochrome P450s). In a l l heme proteins, the b i o l o g i c a l function i s centered on the heme unit and primarily on the iron i t s e l f ( 1-5). Thus the oxidation and spin state of the iron, the nature of the a x i a l ligands, and the protein environment of the heme unit serve as subtle modulators of b i o l o g i c a l behavior. The heme group i s also the p r i n c i p a l o r i g i n of spectroscopic features of these proteins. Both electronic spectra (6-9) and ground-state electromagnetic properties such as quadrupole s p l i t t i n g s i n MOssbauer resonance spectra (10-14), anisotropic g values and hyperfine s p l i t t i n g s i n electron and nuclear spin resonance spectra (15-23) and temperature-dependent magnetic moments (24-29) originate almost e n t i r e l y on the heme u n i t . Consequently, a large f i e l d dedicated to the study o f model heme complexes has emerged i n an e f f o r t to understand the e f f e c t of changes i n the heme unit i t s e l f on these observed properties. These studies are useful i n understanding the properties o f intact heme proteins since isolated heme complexes have electromagnetic proper t i e s very similar to heme units embedded i n proteins. Some of these model heme systems have also been shown to mimic the b i o l o g i c a l a c t i v i t y of intact heme proteins. For instance, model oxo-iron compounds have been found to epoxidize o l e f i n s much l i k e the cytochrome P450s (34, 35). The r e l a t i v e s i m p l i c i t y o f model heme complexes makes i t possible to study the important role of the a x i a l ligands (30-33) i n modulating electronic structure and geometries without the e f f e c t of the nearby amino acid residues present i n the proteins. The insights gained from such studies can help to separately assess the r e l a t i v e importance of the heme unit i t s e l f and of i t s protein environment on the function of intact heme proteins. Up to now most quantum mechanical studies of the ground and excited states of model heme complexes have focused primarily on diamagnetic systems (36), with less frequent treatment of heme systems with unpaired spins (37-42). With the inclusion of a r e s t r i c t e d Hartree-Fock treatment (37, 38) within an INDO formalism parameterized f o r t r a n s i t i o n metals (39, 40, 42), i t i s now possible to calculate the r e l a t i v e energies of d i f f e r e n t spin states o f f e r r i c heme complexes i n an evenhanded fashion at a semiempirical l e v e l . In the work reported here we have used t h i s method i n two types of studies. The f i r s t study i s a systematic investigation of the e f f e c t of changes i n geometry and ligand type on the r e l a t i v e energies of the low-lying spin states and observable properties of eight model f e r r i c heme complexes. This study also represents a test of the c a p a b i l i t i e s of the INDO-RHF method to characterize the lowest lying doublet (S = 1/2), quartet (S = 3/2) and sextet (S = 5/2) spin states of these model f e r r i c heme complexes. The eight complexes chosen a l l have known c r y s t a l structures and include those with varying a x i a l ligands and high-, intermediate-, and low-spin ground

Salahub and Zerner; The Challenge of d and f Electrons ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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Ground-State Properties of Heme Complexes

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states as inferred from magnetic s u s c e p t i b i l i t y measurements (43-50) and g values in electron spin resonance spectra (47-48). They also have known quadrupole s p l i t t i n g s ( A E Q ) from Mossbauer resonance spectra (44, 45, 49, 51-53), a quantity which we d i r e c t l y calculate. In the second type of study, using insights gained from the model compound studies, the active s i t e of the resting state ( i . e . , state of the enzyme when not involved in i t s biochemical cycle) of four heme proteins, cytochrome P450 , metmyoglobin (MMB), cytochrome c peroxidase (CCP), and caualase (CAT) have been characterized. These four proteins belong to d i f f e r e n t classes of heme proteins. P450, CCP, and CAT are oxidative metabolizing enzymes thought to share a similar highly oxidized b i o l o g i c a l l y active state, and MMB i s the oxidized form of an oxygen transport protein with l i t t l e or no peroxidase or monofunctional oxidase a c t i v i t y Q ) . Each of these proteins have f e r r i c resting states which have been characterized by x-ray structure determinations (54-57). Paradoxically, while a number of long-standing questions have been resolved by these structure determinations, new ones are emerging. This study addresses f i v e such s p e c i f i c questions. c

The f i r s t two questions involve properties of the resting state of cytochrome P450 , the only P450 with a known structure (54). The camphor-free resting state i s mostly in a low-spin (S = 1/2) form while the camphor-bound state i s a high-spin (S = 5/2) ferric complex. The x-ray structure (58) reveals, that as previously deduced, the camphor-bound state i s 5-coordinated with a cysteine residue as the single a x i a l ligand and the iron s i g n i f i c a n t l y out of the porphyrin plane. The camphor-free state retains the cysteine ligand, but s u r p r i s i n g l y , a water, and not a second amino acid as previously thought (5), binds to the iron in the d i s t a l ligand binding s i t e . There i s also evidence that t h i s water i s part of an Η-bonded network involving four more water molecules. c

With the insight gained from the x-ray structure, two puzzling aspects of the camphor-free resting state have emerged. One i s the o r i g i n of the low-spin form deduced from observed electromagnetic properties (5). This r e s u l t i s surprising since other f e r r i c heme proteins with H 0 as a s i x t h ligand such as MMB have primarily highspin (S = 5/2) ground states. The other question raised i s : If water i s an a x i a l ligand, as reported in the x-ray structure, why i s no broadening of the ESR spectra from hyperfine interactions observed in 65% enriched 0 HpO (H. Beinert, private communeiation), as i t i s in MMB (59). Since the magnitude of the hyperfine s p l i t t i n g depends d i r e c t l y on the amount of unpaired spin density on the water oxygen atom, the p o s s i b i l i t y that the negative results obtained for P450 could be a consequence of reduced spin density on the water has been investigated. 2

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A l l the remaining questions focus on comparisons of CCP, MMB, and CAT. The f i r s t question addressed i s : Can the differences in the active s i t e of CCP, MMB, and CAT account for the differences in their observed electromagnetic properties? MMB and CCP (55, 56) have been found to have the same heme unit, a f e r r i c protoporphyrin-IX with a water and an imidazole as a x i a l ligands. CAT has a single


342


phenolate group from a nearby tyrosine residue as an a x i a l ligand (57). In addition to the c r y s t a l structures, the nature of the resting states of CCP, MMB, and CAT have been probed by Mossbauer (11-13), temperature-dependent magnetic s u s c e p t i b i l i t y (27-29) and electron paramagnetic resonance (21-23) experiments. The observed properties of both CAT and MMB have been interpreted in terms of a d e f i n i t e high-spin resting state, while the properties of CCP have been interpreted in terms of a thermal d i s t r i b u t i o n of high- and lowspin states (_Π, 29). The determination of the r e l a t i v e energies of the low-lying sextet (S = 5/2), quartet (S = 3/2) and doublet (S = 1/2) states of the active s i t e s of these proteins should lead to a better understanding of the o r i g i n of these properties. The f i n a l two questions raised are the extent to which the active s i t e i t s e l f controls the function of CCP, CAT, and MMB. S p e c i f i c a l l y , we have asked: To what extent can s i m i l a r i t i e s in function between CAT and CCP be understood in l i g h t of their d i f f e r ent active s i t e s ? F i n a l l y , we have asked: To what extent can differences in the function of MMB and CCP be understood in terms of their active s i t e c h a r a c t e r i s t i c s alone? Knowledge of the extent to which the active s i t e can account for function should help to under stand the r e l a t i v e importance of the protein environment around the heme in determining the function of each protein. Methods All calculations were carried out within the approximation of intermediate neglect of d i f f e r e n t i a l overlap (37-42) (INDO-RHF-SCF) which includes parameterization for t r a n s i t i o n metals. A restricted open-shell formalism, developed by Zerner et a l . (37,38), was employed to prevent spin contamination and to make the quantitative evaluation of the r e l a t i v e spin state energies possible. This method has been used successfully to study simple t r a n s i t i o n metal complexes l i k e [ F e C l J * (42), [CUC1J2- (42), and ferrocene (4Ί) as well as larger and more complicated systems l i k e model oxyheme (6M) and carbonylheme (6l_) and model oxyhorseradish peroxidase (62) complexes. Energies of the lowest l y i n g sextet, quartet and doublet states were calculated for each of the heme units studied. The geometries of the complexes were taken from c r y s t a l structures and s i m p l i f i e d to unsubstituted porphyrins. The orientations of the porphyrin macrocycles were such that the pyrrole nitrogens were on the x- and y-axes. The choice of the lowest energy configurations for each state was as follows: Doublet state: Quartet state: Sextet state:

(d

and d

)

3

The sextet state configuration i s unique. The choice of the lowest energy configuration for the doublet and quartet states was confirmed by comparisons of the r e l a t i v e energies of various quartet and doublet configurations, obtained by assignment of unpaired electron(s) to d i f f e r e n t iron d o r b i t a l s , in some representative complexes. It i s also corroborated by a recent detailed study of


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hemin u s i n g the INDO/RHF ( J . P h y s . Chem., i n p r e s s ) .

method

by

Edwards,

Weiner

and

Zerner

The quadrupole s p l i t t i n g ( A E Q ) o b s e r v e d i n Mossbauer resonance o f heme compounds was c a l c u l a t e * from the INDO-RHF e i g e n v e c t o r s . T h i s q u a n t i t y was determined by f i r s t c a l c u l a t i n g the n i n e components (VjJ o f the e l e c t r i c f i e l d g r a d i e n t t e n s o r , u s i n g the a p p r o p r i a t e o n e - e l e c t r o n o p e r a t o r , and c o n s i d e r i n g o n l y the c o n t r i b u t i o n o f the i r o n from a l l i t s f i l l e d o r b i t a l s . The 3> IV j j j > | V j J . These v a l u e s were then used i n the e x p r e s s i o n : k

AE

Q

= 8 ( 1 -

R)Qq[1

+

2

η /3]*

.

( 1 )

where q = V , η = ( V ^ V , J / V * (0 < η < 1 ) , ( 1 - R ) = S t e r n h e i m e r S h i e l d i n g c o n s t a n t , ana Q = n u c l e a r quadrupole moment. The s i g n o f A E Q i s the s i g n o f the l a r g e s t component V . . . V a l u e s o f Q and ( 1 - R ) used i n these c a l c u l a t i o n s a r e 0 . 1 8 7 and O . b o , r e s p e c t i v e l y . i

i

i

R e s u l t s and D i s c u s s i o n Model Heme Complexes. Presented i n T a b l e I a r e the c a l c u l a t e d r e l a t i v e energy d i f f e r e n c e s i n k c a l / m o l f o r the s e x t e t , q u a r t e t , and d o u b l e t s t a t e s o f the model f e r r i c heme complexes i n c l u d e d i n the present study. Also included i n Table I are the calculated quadrupole s p l i t t i n g s U E Q ) f o r the r e l e v a n t s p i n s t a t e , a l o n g w i t h the e x p e r i m e n t a l l y observed v a l u e s o f A E Q and the measured e f f e c t i v e magnetic moments. The r e s u l t s c l e a r l y demonstrate t h a t the ground s p i n s t a t e c a l c u l a t e d f o r each model complex a g r e e s w i t h the one i n f e r r e d from measured e f f e c t i v e magnetic moments. Moreover the energy s e p a r a t i o n s between these ground s t a t e s and the o t h e r two s p i n s t a t e s a r e c l e a r l y consistent with observable electromagnetic properties and help explain their origins. The observed e f f e c t i v e magnetic moments ( 4 3 - 4 6 ) f o r the 5 - and 6 - c o o r d i n a t e d complexes found t o have s e x t e t ground s t a t e s a r e a l l i n the range o f 5 . 9 - 6 y t y p i c a l o f h i g h - s p i n complexes. The c a l c u l a t e d AEQS f o r the h i g h - s p i n s t a t e o f t h e s e complexes a r e a l s o i n good agreement w i t h the e x p e r i m e n t a l v a l u e s known f o r t h r e e o f them ( 4 4 b

46,

5 0 ) .

Both 5 - and 6 - c o o r d i n a t e d h i g h - s p i n complexes have s i g n i f i c a n t s p i n d e n s i t y on the p o r p h y r i n r i n g , 6 0 ? o f which i s on the p y r r o l e nitrogens. T h i s s h o u l d be m a n i f e s t i n h y p e r f i n e s p l i t t i n g s o b s e r v a b l e i n ESR o r ENDOR e x p e r i m e n t s . The u n p a i r e d s p i n d e n s i t y on the a x i a l l i g a n d s i s much l e s s than on the p o r p h y r i n r i n g and g r e a t e r on a n i o n i c than n e u t r a l l i g a n d s . The c a l c u l a t e d r e s u l t s f o r the 5 - c o o r d i n a t e d h i g h - s p i n complexes i n d i c a t e t h a t i t i s d e f i n i t i v e l y more s t a b l e than the d o u b l e t and q u a r t e t s t a t e s by - 3 0 k c a l / m o l and - 1 2 k c a l / m o l , r e s p e c t i v e l y . The 6 - c o o r d i n a t e d h i g h - s p i n complexes e x h i b i t a s i g n i f i c a n t r e d u c t i o n o f the energy s e p a r a t i o n between the s e x t e t and the q u a r t e t s t a t e s ,



2

5.9

5.90

5.92

experimental

6.05

1.12 1.22

0 5 18

TMS0 TMS0

4.5-5.3

3.20 3.5

2 0 25

ClO^

2.09

2.26 3.7-4.7

0 12 12

2.19 2.25

3

a

N" pyridine

0.35

2.31

0 22 19

CN" CN"

Low Spin

3.14 2.7

0 17

3

3-Clpyridine 3-Clpyridine

Intermediate Spin

value of azide complex of MMB and CCP (Reference 53, page 3)

Exp. ( y )

y

e f f

0.12

0 5 18

(NCS)" pyridine

1.01 0.76

0 12 28

2

(SpN0 r

0.44 0.46

A E Q (mm/sec)

0 12 33

Cl""

Cale. Exp.

b

(kcal/mol)

S = 5/2 S = 3/2 S = 1/2

ΔΕ

L

High Spin

"-1

1-2

Table I. Relative Energies of Different Spin States of Model Ferric-Heme Complexes

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A X E ET A K


345

attributable mainly to the presence of the second a x i a l ligand which increases the tendency of the iron atom to move into the plane of the porphyrin. In both 5- and 6-coordinated complexes the energy of the doublet state i s predicted to be too high to play a s i g n i f i c a n t role in determining the observed electromagnetic properties. However, because of the smaller separation of the sextet and quartet states in the 6-coordinated high-spin compounds compared with the 5-coordinated compounds, spin mixing (\f>S) between them should be enhanced. Therefore larger zero f i e l d s p l i t t i n g s and more aniso tropic g values (63) in the ESR spectra should be observed. Intermediate-spin (63) heme complexes are rare and two complexes inferred to have quartet ground states have been included in our studies. As shown in Table I, the predicted ground state of each complex i s a S = 3/2 state in agreement with the spin state assign ment deduced from observed properties. The calculated r e l a t i v e energy of the doublet spin state i s - 20 kcal/mol above the quartet spin state, while the sextet states of [Fe(TPP)(ClO^)] and [Fe(0EP)(3-Clpy)p] are only ~ 1.8 and 2.8 kcal/mol above their respective quartet state. These results strongly suggest that observable properties can best be understood in terms of s i g n i f i c a n t spin-orbit coupling of these two low-lying states, together with the p o s s i b i l i t y of a thermal equilibrium of such spin-mixed states. +

Effective magnetic moment measurements of [Fe(TPP) (ClOi,) ] (47) have yielded values in the range of 4.5-5.3 y at 77-300°K. This temperature dependence and range of values i s consistent with c o n t r i butions from sextet and quartet states with the quartet lower in energy. ESR data (47) for this complex yielded values for g and g| of 4.75 and 2.03, respectively. These results are a t y p i c a l for a high-spin complex and lend further support to the conclusion that the ground state i s a S = 3/2 or a 3/2,5/2 mixture with predominant S = 3/2 character. b

(

Magnetic susceptibility measurements (48) for [Fe(0EP)(3C l p y ) ] , the other intermediate complex studied, y i e l d a range of y between 3.7-4.7 y for the temperature range of 77-294°K. This range of magnetic moments i s also consistent with an intermediatespin or spin-mixed ground state. The EPR spectrum (48) for t h i s complex yielded values for g and g» of 4.92 and 1.97 respectively which are similar to values obtained for [Fe(TPP)(ClO^)]. +

2

e f f

b

The A E Q values calculated for the quartet state of these complexes also agree very well with the experimentally observed values (Table I) for the same complexes. A l l these r e s u l t s taken together are highly suggestive that the S = 3/2 spin state i s the p r i n c i p a l contributor to the ground electronic state of these complexes. For the dicyano and azide-pyridine complexes, our calculated results indicate that in each case a S = 1/2 spin state i s the lowest energy state with the quartet and sextet states much higher in energy. The observed e f f e c t i v e magnetic moments (Table I) of both


346


the dicyano (49) and the azide-pyridine (50) complexes indicate that these two complexes are indeed essentially low-spin at a l l temperatures. The only experimental A E Q measured f o r either of the two lowspin complexes i s f o r the dicyano complex. Our calculated value of 2.31 mm/sec for the S = 1/2 spin state i s i n poor agreement with the experimental value (53) of 0.35 mm/sec. However, the reported experimental A E Q seems to be anomolously low for what i s considered to be a low-spin complex. There i s apparently no experimental A E Q measured for the [Fe(TPP)(N^)(py)] complex. The calculated value or 2.19 mm/sec for the doublet state i s , however, i n good agreement with the experimental values (53) of 2.45 and 2.25 mm/sec f o r CCP-N and MMB-N, respectively, which d i f f e r only by one a x i a l ligand being an imidazole rather than pyridine. This provides further evidence that [Fe(TPP)(N^)(py)] has a doublet ground state. 3

3

An important conclusion from t h i s study of model compounds i s the additional evidence obtained for the key role o f the S = 3/2 spin state i n the chemistry of f e r r i c heme complexes. There are no complexes for which S = 5/2 and 1/2 spin states are close enough to interact without an even greater contribution of the S = 3/2 spin state. Thus, the widely used assumption (64) of high-spin/low-spin thermal contributions to explain observable properties o f heme complexes appears to be incorrect. Explanations involving highspin/intermediate-spin interaction are much more plausible, since small energy separations between these states were found. In general, the r e l a t i v e spin state energies calculated for a l l the model heme complexes studied are consistent with and help explain their observed electromagnetic properties. Thus the INDO-RHF method used appears to be sensitive to the effect of the varying a x i a l ligands and predicts the correct energy order of spin states produced by each of them. The a b i l i t y of the method to predict the patterns of spin state behavior i n these model complexes lends credence to the use made of i t i n the second part of these studies, to further characterize the heme units i n the resting state of four heme proteins. Comparative Studies of Resting State Active Sites of Four Heme Proteins. In this second type of study reported, we have used the x-ray structure of the active s i t e of four heme proteins: cytochrome P450 (54), CCP (55), MMB (56), and CAT (57) s i m p l i f i e d to the f e r r i c porphyrin complexes, shown i n Table I I , to calculate the r e l a t i v e energies and electron and spin d i s t r i b u t i o n s i n their lowlying sextet, quartet and doublet states. çam

As shown in Table I I I , a high-spin ground state i s d e f i n i t i v e l y obtained for camphor-bound 4 5 0 i n which the single a x i a l ligand i s a mercaptide. For camphor-free 4 5 0 , with water and mercaptide as a x i a l ligands at their x-ray structure values, the sextet state i s s t i l l the lowest energy, but the energy separation to the low-spin (S = 1/2) state i s greatly diminished. In the x-ray structure deter mination of the resting state, the value of the Fe-water distance was p

c a m

p

c a m


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Table I I . Ligand Distances (Â, X-Ray) Used for Oxidized Resting State of Four Model Heme Proteins

[MMB] L

[CCP]

H0

2

2

L

1 Fe-0 Fe-L 7 a *Fe

H0

--

Imidazole

Phenolate

2

Imidazole

[CAT]

[P450] H0 2

(SCH3)"

2.40

--

2.02

1.93

1.76

2.32

0.25

0.13

0.13

0.24

1.90

2.24

Extent of out-of-planarity of the iron atom from the mean porphyrin ring plane.

constrained and the exact position of the water was not e x p l i c i t l y optimized. Thus we have considered the consequences of a shorter iron-water distance (2.0 Â) and movement of the iron into the heme plane. In this geometry, a low-spin state i s calculated to predominate. An alternative o r i g i n of the s t a b i l i z a t i o n of the lowspin state comes from the p o s s i b i l i t y that some anionic character i s imparted to the water ligand by i t s postulated interaction with the network of Η-bonded water molecules, seen in the x-ray structure (54). This e f f e c t was simulated by using an OH" as an a x i a l ligand with an Fe-0 bond length of 1.75 A. As seen in Table I I I , in t h i s model of the resting state, the low-spin (S = 1/2) state i s favored by 16 kcal/mol over the high-spin state. While this i s an extreme model for the e f f e c t of Η-bonding, i t does demonstrate that p a r t i a l anionic character of the water ligand could account for the predominant low-spin ground state observed. Table IV gives the spin densities calculated on the water oxygen for P 4 5 0 and for comparison, in MMB. Experimental values of y f f and our calculated results (Table V) indicate MMB i s in a high-spin cam

e

American Chemical Society Library 1155 16th St, N.W. Washington, D.C. of20036 Salahub and Zerner; The Challenge d and f Electrons ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

348

THE CHALLENGE OF d AND f ELECTRONS Table I I I . Origin of the Low-Spin Form of the Resting State of Cytochrome P450

Models for Resting State a

1

(SCH )~

L

H0

H0

S = 1/2

6

S = 5/2

0

L

3

2

2

(SCH )"

Substrate Bound State (SCH )~

b

C

3

3

(SCH )"

(0ΗΓ

—

0

0

15

1.5

16

0

2

d

3

ΔΕ (kcal/mol)

a

Geometry from x-ray structure as shown in Table II (Ref. 54)

b

X-ray structure with Fe-Water distance of 2.00 Â and iron moved into the porphyrin plane

c

X-ray structure with Fe-0H~ distance at 1.75 Â as i t i s in model Fe-0 complexes.

d

Geometry form x-ray structure as in Reference 58.

2

ground state. In i t s c r y s t a l structure geometry, the water oxygen of the high-spin f e r r i c MMB i s calculated to have 0.057e or about 1.1? of the t o t a l spin. For this protein, a barely detectible amount of broadening of the g=2 signal was observed in the ESR spectra in the presence of 0 enriched H 0 (59). By contrast, in both the highspin state and the low-spin state of P450, in i t s c r y s t a l structure geometry, the spin density on the a x i a l water ligand i s much lower than in the corresponding state of MMB. Allowing the Fe-0 distance of the water ligand to decrease, or simulating i t s ionic character by an 0H~, both of which favor a low-spin ground state, somewhat increases the spin density on the oxygen. However i t remains at most about 1/6 that of MMB. Since the broadening in MMB was barely detectable, no measurable broadening of the ESR spectra in 0 enriched water would be expected for either low-spin model of 4 5 0 currently proposed here. These results then account for the absence of such broadening in a manner consistent with the presence of water as an a x i a l ligand in the resting state of 4 5 0 as observed in i t s x-ray structure. 17

2

17

p

c a m

p

c a m

Turning now to comparisons of CCP, MMB, and CAT, the r e l a t i v e energies of the germane doublet, quartet, and sextet spin states have been calculated using the same INDO-RHF-SCF method as for the model complexes and the results are presented in Tables V and VI. The geometries for the resting states of CCP, MMB, and used here were takem dire'ctly from their respective x-ray c r y s t a l


CAT

24.

A X E ET AL.

349


Table IV. Calculated Spin Densities of Oxygen of H 0 Ligands in P 4 5 0 and MMB

a

a

2

cam

MMB

P450 OH"

H0 2

S = 5/2

s

0.01

= 3/2

B

0.16

0.057

0.01

0.030

0.01

0.004

(0.03)

s = 1/2 a

(0.03)

0.0005 (0.004)

Values underlined are for lowest lying state.

^Values in parenthesis calculated for H 0 at 2.00 Â and Fe in the porphyrin plane. 2

Table V.

Effect of Geometry on Resting States of CCP, MMB, and CAT

ΔΕ

D

4h

X-Ray

4h

X-Ray

D

0

0

0

1.5

2.1

2.8

5.7

5.8

6.3

D

4h

(kcal/mol)

S = 5/2

0 (0)

S = 3/2

-1.2 (-2.3)

S = 1/2 AEQ

e f f

a

8.0 (13.0)

b

0

0 (0)

-1.1

2.7 (5.6)

7.7

7.5 (11.4)

(mm/sec)

Cale. Exp. y

CAT

MMB

CCP X-Ray

Exp. ( y ) b

3.20

0.76 1.33

0.72 0.84

4.86

6.00

5.92

a

Values

b

Values in parenthesis with iron moved 0.1 Â further out of mean plane of porphyrin ring.

in parenthesis without d i s t a l water


350

THE CHALLENGE OF d AND f ELECTRONS Table VI. E f f e c t of Ionization State of Axial Ligand i n CCP and Catalase

CCP L L

1

Catalase

H0 Im

H0 Im"

2

2

2

Phenolate

Phenol

ΔΕ (kcal/mol) S = 5/2 S = 3/2 S = 1/2

1.2 0.0 9.2

0 2.4 9.5

Net Charge Fe 1.25 0.11 1 0.18 L •0.55 Porph. Spin Fe 2.79 L1 0.02 L2 0.07 0.12 Porph. a

t

2

2

0 2.1 5.8

b

b

1.35 0.10 -0.63 -0.83

4.26 0.02 0.16 0.56

a

F o r S = 3/2 spin state

b

F o r S = 5/2 spin state

1.37 —

b

4.7 0 12.2

1.28* —

-0.55 -0.82

0.14 -0.42

4.24

2.74

—

0.25 0.51

—

0.11 0.15

coordinates (55-57). However, i n order to examine various geometric e f f e c t s on the spin states of each heme unit, calculations were also carried out at several step-wise regularized geometries, s t a r t i n g from the c r y s t a l geometry of each protein. The e f f e c t s of porphyrin r u f f l i n g and doming were examined by regularizing the porphyrin c r y s t a l geometry to symmetry f o r CCP, MMB, and CAT, while leaving the a x i a l ligands at the same geometry as i n their c r y s t a l structure. Further differences i n the geometries of CCP and MMB were examined by removal of the a x i a l water in CCP and by increasing the out of plane distance of the iron in MMB by 0.1 Â. The calculated r e l a t i v e energies of CCP (Table V) indicate that the S = 3/2 state i s the lowest energy spin state in CCP, with the S = 5/2 and S = 1/2 spin states being -1 kcal/mol and ~9 kcal/mol higher i n energy. Furthermore, the energy ordering and separation of the spin states are rather insensitive to regularization of the porphyrin and a x i a l ligand geometries. The predominance of the quartet state i n CCP appears to be due to a combination of near planarity of the iron and a weak a x i a l ligand. The Fe-water distance in CCP at 2.4 Â i s considerably longer than that i n MMB. Indeed, calculations at the c r y s t a l geometry i n which the water ligand i s


24. A X E ET AL.

351


removed r e s u l t s i n the enhanced s t a b i l i t y o f the q u a r t e t s t a t e o f the complex i n s p i t e o f the 5 - c o o r d i n a t e n a t u r e o f the i r o n ( T a b l e V ) . For MMB ( T a b l e V ) , the s e x t e t s t a t e i s the l o w e s t energy s t a t e f o r the n e u t r a l i m i d a z o l e l i g a n d w i t h the q u a r t e t and d o u b l e t s t a t e s ~3 and ~8 k c a l / m o l h i g h e r i n energy. In c o n t r a s t t o CCP where r e g u l a r i z a t i o n o f the p o r p h y r i n r i n g t o symmetry had l i t t l e e f f e c t ; f o r MMB i t l o w e r s the energy o f b o t h the q u a r t e t and d o u b l e t s t a t e s r e l a t i v e t o the s e x t e t . These r e s u l t s suggest t h a t the enhanced doming o f the p o r p h y r i n r i n g observed i n MMB r e l a t i v e t o CCP i s a f a c t o r i n s t a b i l i z i n g the s e x t e t s p i n s t a t e i n MMB. However, s i n c e the s e x t e t s t a t e i s s t i l l l o w e s t i n energy even when the p o r p h y r i n i s r e g u l a r i z e d t o D j ^ symmetry, the enhanced o u t - o f - p l a n e d i s t a n c e o f the i r o n must be the main c o n t r i b u t o r t o the s t a b i l i z a t i o n o f the s e x t e t s t a t e . T h i s e f f e c t i s v e r i f i e d by the f u r t h e r s t a b i l i z a t i o n o f the s e x t e t r e l a t i v e t o the q u a r t e t s t a t e when the i r o n atom i n MMB i s moved by an a d d i t i o n a l 0.1 Â out o f the mean porphyrin plane (Table V ) . The c a l c u l a t e d r e l a t i v e s p i n s t a t e e n e r g i e s f o r CAT ( T a b l e V) a t the c r y s t a l geometry shows t h a t the the s e x t e t s t a t e i s the most s t a b l e s t a t e w i t h the S = 3/2 and S = 1/2 s t a t e s , r e s p e c t i v e l y , ~2 k c a l / m o l and ~6 k c a l / m o l h i g h e r i n e n e r g y . The c l o s e e n e r g e t i c p r o x i m i t y o f the S = 3/2 s p i n s t a t e i s a r e s u l t o f the s m a l l d i s p l a c e m e n t o f the i r o n atom from the p y r r o l e n i t r o g e n plane. Changing the h i g h l y r u f f l e d p o r p h y r i n m a c r o c y c l e o f CAT t o one o f pure Djj symmetry l e a d s o n l y t o a v e r y s m a l l s t a b i l i z a t i o n o f the s e x t e t s p i n s t a t e o f - 0 . 5 k c a l / m o l r e l a t i v e t o the q u a r t e t and doublet s p i n s t a t e s . Thus, the h i g h l y i r r e g u l a r p o r p h y r i n m a c r o c y c l e i n the c r y s t a l s t r u c t u r e has v e r y l i t t l e e f f e c t upon the r e l a t i v e s p i n s t a t e o r d e r i n g s i n t h i s system. n

In a d d i t i o n t o geometry v a r i a t i o n s , the e f f e c t s o f hydrogen bonding and the r e s u l t i n g i o n i c i t y o f the p r o x i m a l l i g a n d s i n CCP and CAT were s i m u l a t e d by d e p r o t o n a t i o n o f the i m i d a z o l e Ν i n CCP and p r o t o n a t i o n o f the t y r o s i n e oxygen i n CAT. D e p r o t o n a t i o n t o form an Im" l i g a n d i n CCP r e v e r s e s the o r d e r o f the s e x t e t and q u a r t e t s t a t e e n e r g i e s ( T a b l e V I ) . S i n c e t h i s i s an extreme model f o r the p a r t i a l p r o t o n t r a n s f e r t h a t c o u l d o c c u r as a r e s u l t o f the i m i d a z o l e b i n d i n g t o a nearby a s p a r t a t e r e s i d u e i n CCP, the p a r t i a l a n i o n i c n a t u r e c o u l d r e s u l t i n near degeneracy o f the q u a r t e t and s e x t e t s t a t e s . δ

Both the q u a l i t a t i v e and q u a n t i t a t i v e r e s u l t s o b t a i n e d f o r the a c t i v e s i t e s o f the t h r e e p r o t e i n s p r o v i d e an improved b a s i s f o r u n d e r s t a n d i n g the observed e l e c t r o m a g n e t i c p r o p e r t i e s o f the r e s t i n g s t a t e s o f CCP, MMB, and CAT. An i m p o r t a n t a s p e c t o f the p r e s e n t results i s t h a t f o r t h e s e p r o t e i n a c t i v e s i t e s , the s e x t e t and q u a r t e t s t a t e s a r e c l o s e i n energy and the d o u b l e t s t a t e i s s i g n i f i cantly higher. Thus, the dominant contributions to observed p r o p e r t i e s i n these p r o t e i n s a r e expected t o come from the S = 5/2 and S = 3/2 s p i n s t a t e s , which can mix by s p i n - o r b i t c o u p l i n g (63) as w e l l as be i n thermal e q u i l i b r i u m . These r e s u l t s provide a consistent e x p l a n a t i o n o f the e l e c t r o m a g n e t i c p r o p e r t i e s o f the r e s t i n g s t a t e s o f CCP, MMB, and CAT. The a l t e r n a t i v e e x p l a n a t i o n , a thermal e q u i l i b r i u m between s e x t e t and d o u b l e t s t a t e s , w i t h o u t a


352


contribution from the quartet proteins.

state, does not seem possible i n these

These results are p a r t i c u l a r l y important for a correct q u a l i t a t i v e understanding of the the observed properties of CCP. For CCP the experimentally observed magnetic s u s c e p t i b i l i t y ( 2 9 ) and MOssbauer spectra (JM) have been interpreted in terms of a thermal mixing between high- and low-spin states, ignoring any contribution from the intermediate-spin state. This explanation i s contrary to our findings of E ç ^ ~ E3/2 1 / 2 * Measured values of y f f ( 2 9 ) for CCP that are i n the range of 3 . 7 to 4 . 0 y over a temperature range of 7 7 - 2 5 0 ° K can more correctly be understood in terms of a thermal contribution from heavily spin-mixed sextet and quartet spin states. These r e s u l t s also strongly indicate that a re-analysis of the Mossbauer resonance spectra of CCP (JM) as a mixture of quartet and sextet states would also be more appropriate.

Ground-State Properties of Heme Complexes in Model Compounds

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