Download ACTIVE SITES OF HEMOPROTEINS

ACTIVE SITES OF
HEMOPROTEINS
by
Lily Yun Xie and David Dolphin
Department of Chemistry,
Umversity of British Columbia,
Vancouver, BC V6T IZl
Canada
I. INTRODUCTION
The essentially identical iron porphyrin core (heme), attached to a protein through a fifth
ligand, usually a residue group of an amino acid, carries out a wide variety of chemical and
biological processes by combination with different proteins. Hemoproteins function as
carriers of both oxygen and electrons. Biological oxidation of substrates catalyzed by
enzymes containing the heme prosthetic group is a second major function performed by
hemoproteins. For instance, the heme in myoglobin and hemoglobin reversibly bind
dioxygen, while in horseradish peroxidase the heme is oxidized by two electrons producing
water and an oxidized heme protein which then brings about two single one-electron
oxidations of substrates. Cytochromes P-450 which are monooxygenases, on the other hand,
perform all three functions in mat reversible binding of dioxygen is followed by electron
transport and the concomitant reduction of O2 to peroxide. Oxidation of the heme then
occurs upon cleavage of the 0-0 bond to generate water and an intermediate which transfer
oxygen to substrate. It is thus important to understand the structure of the active sites, not
only the oxidation state of the metal ion and the geometry of the complexes, but also the
topology of the heme pocket. Almough high resolution X-ray crystallography provides a
direct visualization of the equilibrium structure of the active site in the solid state,
spectroscopic methods including NMR, resonance Raman and EXAFS are still extremely
valuable for elucidating the dynamics and conformation in solution. The role played by
protein in the catalysis is clearly important; the knowledge of the active site structure has
tremendous impact on the rational design of synthetic models which mimic the activities of
these biological catalysts outside the protein. In this chapter, discussion will be limited to
only a few major hemoproteins.
D. ACTIVE SITES OF HEMOPROTEINS
A. Active site of cytochrome P-450
Cytochrome P-450 enzymes are remarkably versatile monooxygenases and have been
isolated from a variety of sources, including mammalian tissues (e.g. liver), plants, yeast
and bacteria. Substrates that can be oxidized by P-450 include endogenous compounds such
as steroids, lipids etc. and exogenous drugs, pesticides and chemical carcinogens (Gunter
and Turner, 1991; White, 1990). The products of P-450 oxidation may be excretable
metabolites, and initial attention focused on its role in detoxification processes and as an
initiator of chemical carcinogenesis (Black and Coon, 1987; Groves, 1985). The overall
reaction is summarized in equation 1.
The high resolution X-ray crystal structure for the eukaryotic membrane bound
cytochrome P-450 enzymes is currently unavailable. However, these active sites may bear
resemblance to the camphor-metabolizing enzyme P-450cam isolated from Pseudomonas
putida whose X-ray crystal structure is known (Poulos et al., 1985). The prosthetic group
of P-450cam, protoporphyrin IX Fe(III) (heme), is attached to the protein through the
thiolate group of a cysteine residue (Cys357). The heme pocket where the dioxygen and
substrate molecules are bound is composed of entirely lipophilic residue groups of amino
acid such as Leu, Val, Phe (Fig. 1). The architecture of the O2 binding site is closer to that
of hemoglobin and myoglobin than to the more polar peroxidase active sites (Poulos, 1986).
Hydrophobicity of the active site stabilizes the binding of the non-polar hydrocarbon
substrate to protein as well as binding of O2 to iron. Another feature revealed by X-ray
crystallography is that the heme group is buried deep inside the protein pocket; any access
to the active site by the substrate or O2 is only possibly by transient broadening of protein
channels (Poulos, 1986).
The generally accepted mechanism of monooxygenation catalyzed by P-450 (McMurry
and Groves, 1986) is depicted below (Scheme I). While compounds 1 to 4 of the P-450
cycle are well characterized, evidence for the existence of 5 and 6 is still indirect. The
resting state of cytochrome P-450cam is a hexa-coordinate low-spin Fe(II) complex with
H2O as the sixth ligand. Upon substrate binding, water is displaced to give a high-spin
penta-coordinate square-pyramidal complex. A one-electron reduction of Fe(III) to Fe(II) is
facilitated by the change of spin state and precedes the binding of dioxygen to give a lowspin hexa-coordinate complex. The second one-electron reduction yields an iron peroxo
species; subsequent cleavage of the 0-0 bond liberates H2O to give a highly reactive ironoxo species. Substrate is oxidized via oxygen atom incorporation, allowing the enzyme to
Figure 1.
Stereotopic view of the active site of cytochrome P-^SOcim with bound camphor.
Coordinates (entry 2CPP, version of Aug. 1987) for this molecule (Poulos et al., 1987)
were obtained from the Protein Data Bank (Bernstein et al., 1977; Abola et al., 1987) at
Brookhaven National Laboratory.
return to the resting state. The details of O-O bond cleavage, the structure of the resulting
oxygen complex and the mechanism of oxygen atom transfer are less well understood
because the steps that follow the second electron reduction occur too rapidly for any
reaction intermediates to have so far been detected (Andersson and Dawson, 1990). The
description of the assumed bypervalent intermediate has largely been based on analogy of P450 with normal peroxidases (Dolphin, 1985). Furthermore, the observation of P-450
chemistry with exogenous two electron oxidizing agents such as iodosoarenes and alkyl
hydroperoxides implies that the iron-oxo species is perhaps the viable intermediate in the
catalytic cycle, i.e. dioxygen is indeed reduced to a peroxide in the natural sequence
(Groves, 1985). Careful studies of various substrates of P-450 which are of diagnostic
significance suggested that oxygen atom transfer is in fact the result of hydrogen atom
abstraction by iron-oxo species and radical recombination of iron-hydroxo with the organic
radicals. It was also suggested that the organic radical formed by hydrogen abstraction
could not diffuse out of the heme pocket before it collapsed (Groves et al., 1978; Groves
and Nemo, 1983; Traylor et al., 1986).
Designing model compounds that mimic the monooxygenase activity of P-450 without
the benefit of protein has been an active area of research for many years (Meunier, 1992;
Gunter and Turner, 1991). While there still has not been a system mat successfully utilizes
molecular oxygen the way the enzyme does, many model compounds are able to
hydroxylate and epoxidize substrates efficiently via the peroxide shunt route. Metal ions
including Fe (Groves and Nemo, 1983) and Mn (Mansuy et al., 1984), and oxidants
including iodosoarenes RIO (Traylor et al., 1984) and peroxides ROOH (Mansuy et al.,
1982) have been most widely investigated. The ligand in these model systems are primarily
tetraph en yl porphyrin derivatives due largely to the fact that the aryl substituents at the meso
position protect the porphyrin from destruction upon the formation of the p-cation radical
intermediate. Halogenations at ortho positions of the phenyl rings (Traylor et al., 1984;
Traylor et al., 1986) and P positions of the pyrroles provide steric effects which avoid the
formation of μ-oxo dimers and enhance the electrophilicity (oxidizing power) of the
metalloporphyrin entity (Wijesekera et al., 1990).
B. Active site of cytochrome C peroxidase
Cytochrome c peroxidase (CCP), a non-glycoprotein isolated from baker's yeast (Yonetani,
1970), catalyzes primarily the oxidation of ferrocytochrome to ferri cytochrome c by H2O
(eq. 2) (Hewson and Hager, 1979a).
The crystal structures of both yeast CCP (Poulos and Finzel, 1984) and its semistable
oxidized intermediate, compound I (Edwards et al , 1987), are known. In the resting
enzyme the heme is attached to the protein through an imidazole ligand of the proximal
histidine (His 175), and in a highly polar environment consisting of Arg, Trp, Asp, His and
H2O (Poulos and Finzel, 1984) (Fig. 2). Hydrogen bonding occurs between the proximal
His 175 and Asp235, giving the His 175 residue substantial imidazolate character, making it
more electron releasing (Smulevich et al., 1990). The distal His52 is believed to be
involved in acid-base catalysis and Arg48 provides stabilization of negative charge on the
Fe-OOH moiety. The crystal structure of compound I reveals litde difference from the
resting enzyme except that the iron moves about 0 2 Å toward the distal side (Edwards et
al , 1987). This supports an unusually short Fe=O bond which is measured to be 1.67 Å by
EXAFS (Chance et al., 1986). The heme group is located between two protein helices, and
completely covered except for the edges of pyrrole rings A and D; it can be reached by the
oxidant through a well-designed channel leading to the edge of ring D (Finzel et al., 1984).
The size of the protein channel is 6 Å wide and 11 Å long. (Dasgupta et al , 1989). This
unique channel allows for the approach and oxidation of small organic or inorganic
molecules such as guaiacol and ferrocyanide to the d-meso heme edge, but prohibits
macromolecule cytochrome c from approaching the heme; as a result, the oxidation occurs
at a surface site facing the g- meso edge (DePillis et al., 1991).
CCP and P-450 have very different active sites as shown by their X-ray crystal
structures. The heme cavity in CCP is occupied by polar residue groups and the fifth ligand
in CCP is an imidazole which is strongly H-bonded to Asp235. The polar residues on the
distal side and the partial imidazolate on the proximal side may act collectively to cleave the
peroxide bond. On the other band, P-450 has a thiolate on the proximal side and, despite
the lack of polar residues on the distal side, the strongly electron-donating ability of S
appears to be sufficient to cleave the O-O bond heterolytically.
In a freshly prepared sample of the resting wild type CCP, the Fe(ID) atom is pentacoordinate high spin at room temperature over a pH range of 4 3 -7.0 (Dasgupta et al.,
1989; Smulevich et al , 1990); aged CCP contains a mixture of penta- and hexa-coordinate
species. The sixth ligand in this case is possibly a H2O molecule in the high-spin species
Figure 2.
Stereotopic view of the active site of Baker's yeast cytochrome c peroxidase. Coordinates
(entry 2CYP, version of Aug. 1985) for this molecule (Finzel et al., 1984) were obtained
from the Protein Data Bank (Bernstein et al., 1977; Abola et al., 1987) at Brookhaven
National Laboratory.
-
and is OH or distal histidine in the low-spin hexa-coordinate species. A transition takes
place from a penta-coordinate to a hexa-coordinate high-spin ferric form when pH increases
(pKa 5.5) (Dasgupta et al., 1989). The hexa-coordinate ferric CCP is less reactive towards
H2O2 than the penta-coordinate ferric CCP, due possibly to the rate-limiting dissociation of
the sixth ligand (Dasgupta et al., 1989).
The catalytic cycle of CCP involves two single one-electron oxidations of substrate,
generally a protein. It is activated by H2O2 oxidation to a compound with two oxidation
equivalents above the resting state. However, compound I of CCP (known as ES) is clearly
different from that of compound I in horseradish peroxidase (Hewson and Hager, 1979b;
Mauro et al., 1989; Smulevich et al , 1988; Erman et al , 1989; Sivaraja et al., 1989). The
high valent oxyferryl complexes in the catalytic cycle may well have similar electronic
structures, but differ only in the presence of protein radical in the former which likely
resides on the indole ring of the proximal Trp 191 (Edwards et al., 1987). The UV-vis
spectrum of CCP compound I, characteristic of the oxyferryl O=Fe(TV)P, is similar to that
of HRP compound II which is oxidized one equivalent above the ferric state (Coulson et al ,
1971). In contrast to HRP compound I, the MOssbauer and ESR studies of CCP compound
I indicate that the protein-based free radical and Fe(TV) are not magnetically coupled
(Hoffman et al , 1981).
C. Active site of lignin peroxidase
Lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP), both isolated from
Phanerochaete chrysosporiwn, are the two heme peroxidases which are involved in lignin
biodegradation (Tien and Kirk, 1983; Glenn et al, 1983; Glenn and Gold, 1985). LiP exists
as a series of isozymes with molar masses ranging from 38 to 42 kg mol-1. Each isozyme of
LiP consists of a single polypeptide chain and one protoheme IX. Lignin peroxidases, once
oxidized by hydrogen peroxide, function by abstracting one electron from the electron-rich
oxygenated aromatic units of lignin to give aryl cation radicals. Further non-enzymatic
fragmentation of the cation radical leads to the degradation of lignin (Kersten et al., 1985;
Harvey et al., 1985). On the other hand, MnP catalyzes the H2O2- and Mn2+-dependent
oxidation of phenols and other substrates (Glenn and Gold, 1985).
The X-ray crystal structure of lignin peroxidase has recently been solved by Poulos
(Poulos, 1992a) (Fig. 3). The active site of LiP is buried in the protein and the iron center
is not accessible by substrates. A 10 A long open channel in LiP allows H2O2 to approach
the heme, but even veratryl alcohol can best be docked only 6 Å from the heme center
(Poulos, 1992a). In the high-spin penta-coordinate form, the iron is approximately 0.2 Å
out of the heme plane and the iron-nitrogen (pyrrole) distance is 2.05 Å (Poulos, 1992a).
Two calcium-binding sites, one on the proximal side, primarily bound to Ser 177, and
another on the distal side, primarily bound to Asp48, are also located by X-ray
crystallography (Poulos, 1992a). The heme pocket is much like that of CCP, consisting of
polar residue groups such as Asp, Arg and His on both proximal and distal sides (Poulos,
1992a; de Ropp et al., 1991). Instead of having Trp51 and Trpl91 as in CCP, LiP has
Phe46 and Phel93 close to the heme plane. Heterolytic cleavage of the 0-0 bond of
hydrogen peroxide is facilitated by H-bonding on both sides of heme plane (de Ropp et al.,
1991). The vinyl groups of the heme are demonstrated by 1H NMR experiments to take an
in plane orientation which would provide more extensive derealization for the cation
radical in compound I (de Ropp et al , 1991).
The catalytic cycle of lignin peroxidase involving compound I and compound II is
similar to that of horseradish peroxidase which is discussed below. Compound I and
compound II of LiP have been detected by various spectroscopic techniques and are also
similar to those of horseradish peroxidase. At room temperature, the native ferric enzyme is
high-spin penta-coordinate with a vacant coordination site opposite the proximal histidine
(Andersson et al., 1987). At <2°C, a weakly associated ligand, possibly H2O, was observed
(Andersson et al , 1985). However, LiP is unique in that it operates at a low pH optimum
(<3) and exhibits a higher redox potential (Banci and Bertini, 1991). The presence of an
ionizable group in the active site with a pKa <2 has been recently demonstrated by chloride
binding studies (Cai and Tien, 1991). Chloride binds only to the protonated form of LLP.
Figure 3.
Stereotopic view of the active site of lignin peroxidase from P. chrysosporium (Poulos,
1992b).
Formation of compound I, however, requires the deprotonated form of LiP. Binding of
chloride shifts the equilibrium toward the protonated form, thereby inhibiting formation of
compound I at low pH (Cai and Tien, 1991).
The hydrogen bonding of the proximal histidine was recently found to be significantly
less in LiP than in HRP by 1H NMR (Banci and Bertini, 1991). The reduced imidazolate
character observed in LiP destabilizes the oxyferryl species which provide a rationale for
the higher redox potential and lower stability of the compound I of LiP relative to
compound I of the HRP. A weaker axial histidine bond in LiP relative to-HRP has also
been shown by resonance Raman spectroscopy (Andersson et al., 1985).
D. Active site of horseradish peroxidase
Horseradish peroxidase (HRP) is a glycohemoprotein with a molar mass of about
40 kg mol 1. It is isolated from the root of the horseradish plant and catalyzes one electron
oxidations of a wide range of organic substrates, particularly phenols and aromatic amines
(Hewson and Hager, 1979a), and is characteristic of many plant peroxidases.
The catalytic cycle of horseradish peroxidase (Andersson and Dawson, 1990; Dawson,
1988) is depicted in Scheme II.
Efforts to obtain an X-ray crystal structure of HRP have not been successful
(Brathwaite, 1976; Aibara et al., 1981). Considerable emphasis has been placed on the use
of spectroscopic methods (Thanabal et al., 1987; Smulevich et al., 1990; Terner et al. 1989)
and sequence homology to CCP (Sakurada et al., 1986) in attempting to define an active site
structure. 1H NMR experiments have been particularly useful in identifying the catalytically
significant amino acid residues which are involved in the H-bonding network on both the
proximal and distal sides of the heme plane (Thanabal et al., 1987). The heme prosthetic
group in horseradish peroxidase is attached to protein through the imidazole of His 170
which remains coordinated throughout the catalytic cycle. The heme edges are buried in the
protein as indicated by the formation of the 5-meso alkylated product by pbenylhydrazine
derivatives instead of the usual N- alkylated product as in the case of cytochrome P-450.
The highly oxidizing iron center is completely inaccessible to substrates (Ator and Ortiz de
Montellano, 1987; Ortiz de Montellano1 1987). The heme pocket is composed of polar
residue groups of amino acid such as Arg38 and His42 capable of forming hydrogen bonds
(Sakurada et al., 1986). Arg38 and His42 are believed to play important roles in cleavage of
the 0-0 bond of peroxide for the generation of compound I and for its stabilization. The
imidazole ring of the distal bistidine is protonated upon H2O2 oxidation, the imidazolium
proton forming a H-bond with and polarizing the 0-0 bond Cfhanabal et al., 1988). Studies
show that imidazole of the proximal histidine exhibits two coordination modes. Both 1H
NMR and resonance Raman spectroscopy show hydrogen bonding interactions between the
proximal Hisl70 and nearby groups (Thanabal et al., 1987; Smulevich et al., 1990; Terner
et al., 1989). The proximal imidazole ligand is neutral when the heme is pema-coordinate,
but deprotonates to become a negatively charged imidazolate ligand when the heme becomes
hexa-coordinate in compound I and compound II (Thanabal, 1988). The negatively charged
imidazolate donates electron density to iron, and from iron to the 0-0 bond. This 'pushpull ' mechanism involving hydrogen-bonding networks on bom the proximal and distal side
of the heme explains the ease of the heterolytic cleavage and stability of the resulting
.
O=Fe(IV)P+ and O=Fe(IV) complexes. The imidazolate character in horseradish
peroxidase is also seen in other peroxidases in varying degrees (Finzel et al., 1984;
Smulevich et al., 1990; Banci and Bertini, 1991). The degree of deprotonation or
imidazolate character is, however, seemingly related to the stability of the ferryl oxygen
complexes (Morrison and Schonbaum, 1976; Schonbaum, 1982) and thus to the redox
potential of the corresponding peroxidases (Band and Bertini, 1991).
The Fe(III) ligation of the resting state of horseradish peroxidase is characterized as
penta-coordinate high spin (Smulevich et al., 1990). Upon oxidation by hydrogen peroxide,
a very reactive intermediate called compound I which is two oxidation equivalents above the
native ferric state is formed. Compound I is a low-spin hexa-coordinated Fe(IV) porphyrin
p-cation radical formulated as O=Fe(TV)P+ . The presence of a p -cation porphyrin radical
in compound I has been validated by several experimental methods including UV-vis
(Dolphin et al., 1971), ESR (Roberts et al., 1981; Rutter et al., 1983) and NMR
spectroscopies (La Mar et al., 1981). Magnetic susceptibility data (Theorell and Ehrenberg,
1952) have been interpreted as a ferromagnetically coupled combination of a low-spin
Fe(IV) (S= 1) and a porphyrin x-cation radical (S= 1). Compound I readily undergoes a one
electron reduction by organic substrates to compound II which is characterized as an
O=Fe(IV)P, supported by compound II model studies (Chin et al., 1980). A second
substrate molecule can be oxidized by compound II which allows the enzyme to return to
the resting state. Unlike the organic radical formed in P-450 monooxygenase, substrate free
radicals formed during the HRP cycle readily diffuse into solution. Oxygen atoms when
incorporated into the oxidized product are not from the H2O2 oxidant, but from the solvent
or O2 reacting in a later stage of the reaction steps (Ortiz de Montellano et al., 1987).
Although horseradish peroxidase catalyzes some oxygen-incorporating reactions including
the expoxidation of styrene, it requires the presence of a cosubstrate or co-oxidant which is
capable of forming a radical by a one-electron oxidation of horseradish peroxidase; the
peroxy radical formed from the cosubstrate was found to be responsible for the styrene
oxide formation (Ortiz de Montellano and Grab, 1987).
E. Active site of catalase
Most mammalian catalases except HPII from E. Coli and Neurosporo crassa contain iron
protoporphyrin DC as the prosthetic group. Beef liver catalase is shown by X-ray
crystallography to contain four identical subunits. Catalase protects cell from the toxic
effects of H2O2 by catalyzing a disproportionation reaction (eq. 3) (Hewson and Hager,
1979a).
The heme site of beef liver catalase in one subunit is accessible by a channel 30 Å long
as determined by X-ray analysis. The channel starts with the polar residues Arg, Asp, Gln
and Lys at the surface and ends with the hydrophobic residues Val, Pro, De, Leu and Phe
on the heme distal side (Murphy et al., 1981). The aromatic rings of Phel60 and His74 on
the distal side stack parallel to the porphyrin plane. Distal Asnl47 and His74 play important
roles in substrate binding both in the formation and reduction of compound I. Specificity is
largely determined by the ability of a substrate capable of forming H-bonding interactions
with these two residues (Fita and Rossmann, 1985a). Both propionyl groups are involved in
Figure 4.
Stereotopic view of the active site of beef liver catalase. Coordinates (entry 7CAT, version.
of Nov. 1984) for this molecule (Fita and Rossmann, 1985b) were obtained from the
Protein Data Bank (Bernstein et al., 1977; Abola et al., 1987) at Brookhaven National
Laboratory.
a complex network of electrostatic and hydrogen bonding interactions. Unlike peroxidases,
the fifth ligand of catalase is the phenol ring of Tyr357. As determined by X-ray
crystallography, in beef liver catalase, the phenyl plane is tilted 42° inwards to the heme
plane with an iron - oxygen distance of 2.2 Å (Vainshtein et al., 1986) (Fig. 4). This
suggests that the phenolic side chain is deprotonated and possess a localized charge (Fita
and Rossmann, 1985a). Arg353, between Tyr357 and His217, is involved in H-bonding
interaction and is suggested to play an important role in the catalytic function (Fita and
Rossmann, 1985a).
The reaction cycle of catalases (Scheme III) begins with die high-spin ferric state which
reacts with one molecule of hydrogen peroxide to form compound I. Second hydrogen
peroxide reduces compound I in one step to the resting state with the concomitant
generation of dioxygen (Andersson and Dawson, 1990). The second reaction has been less
well studied.
Formation of compound I is similar to horseradish peroxidase and the radical is also
located at the heme ring (Dolphin et al., 1971). Catalase prefers two electron donors in the
reduction of compound I to the resting enzyme, whereas peroxidase prefer one electron
donors, with the reduction of compound I occurring in two distinct reaction steps via
compound II. In the presence of two-electron donors, e.g. H2O2, the formation of
compound IIis never seen. Catalase also has a high degree of specificity and shows a
strong preference for H2O2 in the reduction. Alkyl or acyl hydroperoxides do not reduce
compound I (Fita and Rossmann, 1985a). However, N-demethylation analogous to reactions
catalyzed by P-450 has been observed in some cases (Kadlubar et al., 1974).
F. Active site of globins
Hemoglobin is the oxygen carrier in vertebrate blood. It consists of two a and two b
subunits. The active sites of the a and b subunits are both hydrophobic but not identical and
both bind heme. Myoglobin is a similar protein consisting of a single chain and one heme
group. It stores O2 in muscles and releases it to the mitochrondrion for the production of
ATP (Ten Eyck, 1979).
The resting state of Hb and Mb is an Fe(II) penta-coordinate high-spin complex which
binds O2 reversibly to form dioxygen complexes which are hexa-coordinate Fe(II) low spin.
X-Ray crystallography of human dexoyhemoglobin confirmed that the proximal ligand is
His87 and dioxygen binds to the iron in a tenninal bent fashion. Binding of dioxygen is
stabilized by an H-bonding interaction between the terminal oxygen and the distal His58
(Phillips and Schoenborn, 1981). The H-bond distance in the a-subunit is similar to that in
MbO2 while that in the B-subunit is longer, indicative of a weaker H-bond. The Fe atom is
displaced significantly towards the proximal His87 in the deoxy form, but moves into the
porphyrin plane upon oxygenation (Perutz et al., 1987) (Fig. 5).
The interaction of O2 with Mb is a simple bimolecular reaction and the oxygen affinity
of Mb is independent of environment. However, the reaction of O2 with Hb is cooperative
and influenced by both heterotropic and homotropic ligands. The cooperativity observed in
various hemoglobins has been extensively studied (Perutz et al., 1987).
Figure 5.
Stereotopic view of the active site of human deoxyhemoglobin: a-chain. Coordinates (entry
3HHB, version of Mar. 1984) for this molecule (Fermi et al., 1984) were obtained from the
Protein Data Bank (Bernstein et al., 1977; Abola et al., 1987) at Brookhaven National
Laboratory.
Despite the fact that both myoglobin and hemoglobin have been optimized to store and
transport oxygen, they are also capable of catalyzing redox reactions under appropriate
conditions (Mieyal, 1985). The fifth ligand in these proteins cannot form hydrogen bonds
with nearby residues which makes it a poor electron-donating ligand unlike the proximal
histidine in peroxidases. The lack of a strongly polarizing arginine residue on the distal side
also renders dioxygen bond cleavage much less facile compared to the peroxidases. The
reaction of Hb and Mb with H2O2 produces an oxyferryl species and a protein radical
similar to that found in CCP but in mis case the radical resides on a tyrosine residue
(Yonetani and Sihleyer, 1967). Although the active center is accessible to substrate, 70% of
the oxygenated product in styrene epoxidation was found to contain oxygen from O2 rather
than H2O2. A peroxy radical on Tyr42 formed by O2 addition to the phenyl radical is
perhaps responsible for the formation of styrene oxide (Ortiz de Montellano, 1987).
III. SUMMARY
All the hemoproteins discussed above are involved in interaction with O2 and/or H2O2 of
some type. They are closely related in their structures but each performs a different
function. The key feature of heme protein catalysis is their ability to form an oxyferryl
species by cleavage of the 0-0 bond formed either by dioxygen or H2O2 interaction. The
mechanism for the formation of mis oxyferryl or compound I varies from one system to
another, and is governed largely by the nature of the proximal ligand, and the polarity of
the proximal and distal heme environment. In peroxidases, the proximal ligands are
histidine. The imidazole ring is not a very effective electron donor in the absence of
hydrogen bonding to other groups; therefore, subtle difference in the amino acid sequence
and polypeptide folding can dramatically alter the hydrogen bonding ability to imidazole.
The Strengm of the imidazole H-bonding to neighboring residues has a significant influence
on the imidazolate charcter of the proximal histidine. The heme environment in peroxidases
are invariably polar. In addition, the imidazole character is also reflected in the oxidizing
power of the enzymes. The greater the negative charge on the imidazole, the more stable the
oxyferryl species. The imidazole remains a neutral ligand in hemoglobin and myoglobin
because the hydrogen bonding effect to imidazole is absent. The lack of electron-donating
effects from nitrogen to iron makes Hb and Mb poor peroxidases. The fifth ligand effect is
dominant in P-450 and catalases, with thiolate from cysteine and phenolate from tyrosine
respectively. Thiolate and phenolate are much better electron-donating ligands than
imidazole, which may account for the readiness of heterolytic cleavage of the O-O bond in
these two proteins even though the residue groups on the distal sides are entirely
hydrophobic. Partial imidazolate character alone is perhaps not sufficient to cause the
cleavage of the O-O bond. Therefore, nature places polarizing residues on the distal side to
assist the heterolytic cleavage.
Compound I, two oxidation equivalents above resting enzyme, is believed to be the
common reactive intermediate formed in hemoprotein catalysis, having two basic types of
structures O=Fe(IV) porphyrin cation radical (P-450, HRP, LiP and Cat) or protein radical
(CCP on Trp, Hb and Mb on Tyr). The structures of these compounds I with different
proximal ligands (S, 0, N coordination) have been studied by various spectroscopic
techniques including X-ray structure for CCP (Edwards et al., 1987). The 1.67 Å bond
length of Fe-O measured by EXAFS is consistent with an Fe=O double bond and an iron
oxidation state of TV. EPR and Mossbauer spectroscopies are very useful for identifying
location of the radical.
It is not surprising that model systems free of protein lack of the substrate binding
specificity imposed by the protein structures; therefore, it is still not possible to achieve the
catalytic specificity obtained by heme proteins, even though greater turn-over-rates may be
observed (Traylor et al., 1984; Traylor et al., 1985).
ACKNOWLEDGEMENTS
This work was supported by the Canadian Natural Science and Engineering Research
Council. Figures were generated on a Silicon Graphics workstation using Biosym InsightII
software.
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