Nod" 3 Hod NHoa*'2 Hod NH2~
4.7 ELEMENTARY STEPS IN BIOCATALYTIC OXIDATION REACTIONS .1 Introduction
4.7.2 Electron transfer reactions
Without biological electron transfer reactions (also called reduction/oxidation or redox reactions) life would not exist. Well-organized electron transfer reactions in a series of membrane-bound redox proteins form the basis for energy con- servation in photosynthesis and respiration. The basic reaction is simply the transfer of electrons from the donor to the final electron acceptor. Perhaps the best example of these redox reactions, their importance for living organisms, and the nature of the different type of biocatalysts that are involved is the respiration chain present in the membranes of mitochondria. The membrane-bound nature of this electron transport chain, supporting electron transfer from N A D H to 02 as
4 -- BONDING AND ELEMENTARY STEPS IN CATALYSIS 189 a)
HHH
( 5 6 6CHr-
.r162
CH2- Oi .P" OI HHH 'O.
|
"30 NI-~N ,
!
O ,
|
flavin mononucleotide (FM N)
NH2
o_
"O'P:O ,o
HO OH
flavin adenine dinucleotide (FAD)
b) CH2 =CH CH3
H3C CH=CH2
H 3 C ~ - - C H 3
CH2 CH2
I I
CH2 CH2
I I
COO COO
heine
b
Iron-protoporphyHn IX
Fig. 4.75. Schematic drawings of the structures of the (a) flavin (FMN and FAD) and (b) heme cofactor for biocatalytic oxidations in their inactive resting states.
the terminal electron acceptor, is essential. This is true because protons are translocated across the membrane as electrons are passed along the electron transfer chain, generating a transmembrane proton gradient and an electrostatic potential gradient, which can subsequently be used to generate ATP, the im- mediate energy source for constructive cellular processes. Figure 4.76 presents an overview of the respiratory chain, and illustrates especially the nature of the electron-carrying redox proteins involved, which contain a variety of different types of cofactors.
The main point to stress from a biocatalytic perspective is the unique nature of the flavin cofactor in these types of electron transfer reactions. Whereas all other
NADH NAD +
~ M N - ~ F e S } ~ / / ~ ~ - ~ B e m e herne ~_~~h~e
r )
herne heme
Fe - ~ Fe
cyta cyta 3
complex I complex III complex IV
(oi)
Fig. 4.76. Schematic representation of the respiratory electron transport chain. Note the presence of a variety of different cofactors.
190 4 - - B O N D I N G A N D E L E M E N T A R Y STEPS I N C A T A L Y S I S
cofactors exclusively accept and donate either only one (e.g., hemes, FeS clusters) or two (NAD(P)H) electrons at a time, the flavin cofactor can do both. Thus, flavin-containing electron transfer proteins provide the natural link between one- and two-electron transfer proteins. This is also elegantly illustrated by the electron transport processes in the respiratory chain, where an FMN-type flavin cofactor forms the link between NADH and an FeS cluster.
Finally, it is also important to mention that electron transfer by proteins is a process that has been studied in great detail recently. The prevailing hypothesis at present is that electron transport "through bonds" in the protein is more efficient than electron transport "through space". Regardless of the precise mechanism, however, electrons can be transported at reasonable rates over relatively long distances (up to 15 to 20 ~), and thus from one protein cofactor to another.
4.7.3 Heme-based peroxidases
Heme-based peroxidases are biocatalysts that perform the oxidative conversion of a wide range of substrates by means of formation of a remarkably reactive electrophilic species; the general catalytic cycle is schematically depicted in Figure 4.77.
Upon interaction of hydrogen peroxide with the Fe B+ metal centre that is present in the heme cofactor of the enzyme in the resting state, cleavage of the
H 2 0 + A +* PorFe I, H202
A
+ 2 H* rate
(PorFeO) 2§ ( or FeO)
compound II c o m p o u n d I
A +" A
H20
o .-II ~ , ,_//,F.;NZ
Fig. 4.77. Catalytic cycle of heme-based peroxidases.
4 -- BONDING AND ELEMENTARY STEPS IN CATALYSIS 191
H I N His- \ \
N.
" H - - : O - - H . . . . H*B
/
~ F e ~
(;') I
/ \ H i s B . . . . H
Fig. 4.78. Role of the distal histidine in the "pull" mechanism for the cleavage of the dioxygen bond and creation of the high-valent iron-oxo porphyrin ~ cation radical (Compound I) in peroxidases.
dioxygen bond is achieved. This leads to formation of the so-called compound I.
The exact nature of this compound I has been deduced from studies with simple chemical model systems, and its existence has also been proven directly in several heme-based peroxidases, such as for example the most widely studied peroxidase, horseradish peroxidase. Compound I can best be described as an Fe(IV)-oxo porphyrin ~ cation radical, in which the Fe is referred to as high- valent state because it is in an oxidation state above the normal Fe(II) or Fe(III) valency. Originally the Fe might even be present in an Fe(V) state, but this has never been observed. The supposed Fe(V) (if formed initially) instead seems to abstract an electron from the porphyrin ring system of the heme, thus leading to formation of the Fe(IV)-oxo porphyrin ~ cation radical intermediate depicted schematically in the catalytic cycle of Fig. 4.77.
This high-valent iron-oxo species is among the most powerful oxidizing species known in biology. Formation of Compound I from the FeB+-heme con- taining resting state and H 2 0 2 in the active site of peroxidases is assisted by the so-called distal histidine, as depicted in Fig. 4.78. This mechanism is often referred to as the "pull" mechanism for dioxygen bond cleavage by peroxidases.
The proximal histidine, acting as an axial ligand to the Fe centre, is not involved in this mechanism for Compound I formation.
The highly electrophilic Compound I intermediate is able to catalyze the one-electron oxidation of a wide variety of substrates, leading to a wide range of different types of conversions. Examples of reactions catalyzed are the dimeri-
192 4 ~ B O N D I N G A N D E L E M E N T A R Y STEPS I N C A T A L Y S I S
zation of phenols through formation of phenolic radicals, formation of coloured dimeric reaction products, polymerization reactions a n d / o r the formation of products resulting from the reaction of substrate radicals with solvent molecules such as H20, or from dismutation of radicals to give two-electron oxidized reaction products. In addition, the strong oxidizing power of peroxidase Com- pound I intermediates might be useful for bleaching reactions of interest for the paper and detergent industries. Since peroxidases show a wide substrate specificity and u s e H202 as a clean oxidant to create the reactive Compound I species, industrial interest in these peroxidases is no surprise. In addition, their reactions may be of interest commercially, since they may affect taste and result in browning of fruits and vegetables through polymerization reactions.
Once Compound I has catalyzed the one-electron oxidation of a substrate, the porphyrin cation radical is "neutralized" and the so-called Compound II is formed (Fig. 4.77). This Compound II, perhaps upon protonation of its oxo atom, is still a powerful oxidizing species, and can catalyze the one electron oxidation of a second substrate molecule, leading also to formation of a molecule of water and conversion of the heme moiety to its Fe B+ resting state. Because the oxidative power of Compound II is lower than that of Compound I, substrate oxidation by Compound II is the rate-limiting step in peroxidase biocatalysis.
4.7.4 Monooxygenases
The biocatalysts capable of performing monooxygenation reactions are of special interest from a catalytic point of view, since in these reactions transfer of one of the oxygen atoms from molecular oxygen to the substrate occurs. The overall reaction catalyzed by monooxygenases is
S + 02 + NAD(P)H + H + ~ SO + H20 + NAD(P) §
Monooxygenase reactions can lead to a wide range of conversions, such as hydroxylations, epoxidations, heteroatom oxygenations, heteroatom dealkyl- ations, and oxidative dehalogenations. Some of the monooxygenases not only catalyze a wide range of reactions under relatively mild conditions, but they also do so in a stereo- a n d / o r regiospecific way. In addition, in some cases these monooxygenases show an extremely broad substrate specificity. For example the mammalian cytochromes P450 are a family of enzymes able to convert over 300,000 different substrates. For all these reasons, the enzymes capable of catalyzing monooxygenase reactions are of special industrial interest.
From a biocatalytic point of view the most interesting question to be answered is what enables these enzymes to catalyze the monooxygenation of even relatively inert chemical substrates, such as benzene derivatives, under mild conditions. To answer this question several examples of monooxygenases, with different cofactors used to perform the job, will be discussed.
4 ~ B O N D I N G A N D E L E M E N T A R Y STEPS I N C A T A L Y S I S 193 4.7.4.1 Flavin-dependent monooxygenases
Flavin-dependent monooxygenases function through a reaction cycle that is schematically presented in Fig. 4.79. These enzymes perform their catalytic reaction through the generation of a reactive form of the flavin cofactor, formed by 2-electron reduction of the flavin followed by its reaction with molecular oxygen. As a result, the so-called C(4a)-peroxyflavin intermediate is formed, whose electrophilic reactivity is further increased by protonation of the distal oxygen of the peroxide to yield the C(4a)-hydroperoxyflavin form of the cofactor (Fig. 4.80).
This C(4a)-hydroperoxyflavin intermediate is the active form of the cofactor in a monooxygenation reaction, leading to the hydroxylation of aromatic sub- strates. The reaction mechanism is schematically depicted in Fig. 4.81. Table 4.3