Catalytic Reactions of Radical Enzymes

L.A. Eriksson (Editor) Theoretical Biochemistry - Processes and Properties of Biological Systems Theoretical and Computational Chemistry, Vol. 9 9 2001 Elsevier Science B.V. All rights reserved

145

Chapter 4

Catalytic Reactions of Radical Enzymes Fahmi

Himo a

and Leif A. Eriksson b

aDepartment of Physics, Stockholm University, Box 6730, 113 85 Stockholm, Sweden. [email protected]. bDepartment of Quantum Chemistry, Box 518, Uppsala University, 751 20 Uppsala, Sweden. [email protected]

1. Introduction

The number of enzymes discovered to harbour and employ a metastable radical site for the catalytic activity has steadily increased over the past decades [1]. Besides 'pure' radical enzymes, i.e., systems that use a stable radical for the catalytic action at the active site, theoretical studies indicate that radical intermediates are also employed in several other systems - see e.g. the chapter by Siegbahn and Blomberg. In addition, many key reactions in biology make use of radical forms of cofactors such as quinones in photosynthesis (see chapter by Wheeler), the vitamin E controlled quenching of lipid peroxidation or the various catalytic mechanisms involving radical forms of coenzyme B12 (see chapter by Radom et al). The form in which the radical nature is stored and employed hence differs significantly from system to system, and the aim of the present chapter is to give a flavour of some of these aspects with key focus on radical enzymes. Radical enzymes are, like radical reactions in general, usually characterised by high turnover for the catalytic processes. It is hence rather difficult to study these reactions experimentally in order to gain direct insight into the mechanisms. In addition, several of the enzymes are membrane bound or anaerobic, why the determination of the crystal structures of many of these has been a formidable task. Most of the mechanistic proposals have thus been based on mutagenesis experiments (site specific exchange of an amino acid), kinetic measurements and isotope effects, and studies of inhibitor 'by-products'. Despite their high reactivity, radical systems do, however, have one advantage over non-radical systems - the existence of unpaired spin. The unpaired spin leads to interaction between the magnetic moments of the

146 unpaired electron and the magnetic nuclei in the sample; interactions that are observable via electron spin/paramagnetic resonance techniques (ESR, EPR; as well as more advanced related methods such as ENDOR and TRIPLE spectroscopies). These so-called hyperfme parameters can be used to identify the radical center as well as to single out protonation states and geometric conformers of the different radicals observed, thus providing further insight into the mechanis ms. The radical hyperfine coupling constants (HFCCs) can also be computed with high accuracy. Most commonly, we report isotropic HFCCs (Ai~o, the traces of the diagonatized 3x3 hyperfme interaction tensor), also known as Fermi contact terms, that essentially are overlap integrals times unpaired spin population at r=0 (i.e. in each magnetic nucleus). You will however also see anisotropic data r e p o r t e d - these are the remainder of the diagonalized HFCC tensors once the trace has been removed, usually denoted Txx, Tyy and Tzz (if the full diagonal terms are reported, i.e., Ai~o + Txx etc, these are labelled Axx etc). The anisotropic couplings describe the non-spherical/spatial distribution of the unpaired spin surrounding each nucleus, and are computed using dipole-dipole integrals. Experimentally, also the so-called g-factors are reported. These determine at what magnetic field or frequency we might find the center of the spectrum, and also provide important information about the nature of the radical in question. Computationally, however, g-factors are much more difficult to compute than hyperfine couplings - to date only a few reports are available on biologically relevant systems- and we will throughout assume the free electron value for g, 2.0023, in our reported data. One of the advantages with theory is that we are able to test a large set of mechanistic proposals, in order to determine which that from an energetic point of view are the most likely ones. The mechanisms discussed herein are thus mainly those representing the energetically most favourable paths for model systems in vacuum (or in a dielectric cavity). Theoretical data should hence not be regarded as 'the truth' but rather as one more piece of information towards a complete understanding of how the various enzymes function, to complement the various experimental studies. We should also emphasize that for each of the systems discussed herein, several additional mechanisms/paths have been explored but discarded based on unreasonable energetics, spin distributions that do not comply with experimental findings, and similar. Using computed HFCCs, geometric structures, and energy surfaces we can hence construct a most probable reaction path for the catalytic mechanisms, and we will herein report on detailed theoretical studies of model systems to Galactose oxidase (GO) [2], Pyruvate formate-lyase (PFL) [3,4] and Ribonucleotide reductase (RNR) [5,6]. To finally verify the suggested mechanisms, the full enzymes (or as much as possible thereof) need to be taken into account, as well as temperature, dynamics, solvents, etc. We are presently witnessing the arrival of such approaches, combining different methodologies

147 into e.g. the QM/MM methods, or DFT-MD. We also refer the interested reader to some of the many excellent reviews available, where the experimental fmdings on the above systems are summarized [1,7,8].

2. Methodology All systems outlined in the present work have been studied using the hybrid Hartree-Fock- Density Functional Theory functional B3LYP [9]. Geometry optimizations and frequency calculations are throughout performed using a split valence basis set; normally 6-31G(d,p) or LANL2DZ (the latter in case the system contains transition metals) [10]. In order to obtain reliable energetics and spin properties, B3LYP calculations are subsequently performed on these optimized structures, using larger triple-zeta plus multiple polarization function basis sets, in some cases also including diffuse functions, i.e. 6-311(+)G(2df, p) or Lanl2DZ(+)(2d,2p). In addition, the energetics are corrected for zero point vibrational effects, as obtained using the smaller (geometry optimization) basis set. All calculations are performed using the Gaussian 94 and Gaussian 98 suites of programs [11,12]. When investigating enzymatic reactions using quantum chemical techniques, it is not possible to include the full enzyme. Instead, rather crude approximations need to be taken with respect to the size of the model employed. At present, such 'all-quantum' models are limited to the range of 30-50 atoms. In the present work, different criteria have been employed in order to determine the sizes of the individual model systems. In Table 1 we list the S-H bond dissociation energy for the amino acid cysteine, commonly employed by nature in radical enzyme reactions. As seen, extending the description of the cysteine side chain beyond a methyl group gives very minor improvements in the S-H bond strengths, compared to using the full amino acid. On the other hand, using the smallest possible model, HS-H, the BDE deviates by ca 6 kcal/mol from the amino acid and is obviously not a suitable model. Table 1. Effects of model system size on S-H bond strength in cysteine (kcal/mol), computed at the B3LYP/6-31 l+G(2d,2p) level. System HS-H CH3S-H CH3CH2S-H NH2(COOH)CHCH2S-H

S-H bond strength 87.1 81.9 81.9 81.7

Another criterion for choosing the models is based on the radical hyperfine structures. As mentioned above, the hyperfine properties can also be employed to single out protonation states and geometric conformers of the different

148 radicals observed, thus providing further insight into the mechanisms. Radical hyperfme couplings arise from interaction between the magnetic moments of the unpaired electron and the nuclei, and may be divided into two sets; the isotropic components (the trace of the diagonalized full 3x3 hyperfine interactions tensor), and the anisotropic 'remainder' of the diagonalized tensor. For detailed accounts on the theoretical evaluation of radical hyperfme tensors, the reader is, e.g., referred to references [ 13] and [14]. One example illustrating this particular aspect of model construction is the glycyl radical present in PFL and anaerobic RNR. In this case, the amino acid side chain consists of a hydrogen atom only. Removing one of the I-I~'s (i.e., the side chain) hence leads to the formation of a backbone-centred radical, in which the unpaired spin may delocalize out into the backbone rather than (as in most other cases) remaining localized on the side chain. In Table 2 we list some computed HFCCs for a series of models of the radical center in the glycyl radical. In this case it is clear that using a small model or the amino acid alone is not sufficient, but that we need to use the more extended form in order to accurately describe the distribution of the unpaired spin, the radical properties, and thus provide an accurate model for the systems reactivity. Table 2. Ca and Ho~ isotropic HFCCs (gauss) for a sequence of model systems, from methyl radical to extended glycyl, computed at the B3LYP/6-31 l+G(2d,2p) level. System CH3 CH2-CH3 NH2-CH2 CH2-CHO NH2-CH-CHO NH2-CH-CO-NH2 CHO-NH-CH-CHO CHO-NH-CH-CO-NH: CH3-CHO-NH-CH-CO-NH-CH3 Exp. E. Coli PFL [ 15] Exp. E. Coli anaer. RNR [ 16]

Aiso(1H~) -22.8 -21.9 -10.1 -17.6 - 11.5 - 14.2 - 13.8 - 16.4 - 16.4

&so(13C~) 25.9 29.2 44.4 19.3 7.3 11.5 11.7 15.5 15.3

(-)15 (-)14-15

16-21 :5-21

In addition to the above procedure to determine the smallest possible (yet accurate) models, we have also applied a few additional criteria. The computations are normally conducted in gas phase; i.e. all interactions with the surrounding medium (solvent and remaining enzyme) are neglected. In certain cases, however, a dielectric medium based on a polarized contiuum model (PCM) [17], is introduced. The protein backbone is for the most part excluded, assuming the reactions to essentially be localized to the side chains. However,

149 when two neighbouring amino acids are involved in the same reaction, the backbone connecting these is retained in order to mimic the restricted motion of the side chains relative to each other. We furthermore try to use charge neutral models as far as possible, based on convergence properties of charged species in vacuum. Apart from this, the reacting groups are always allowed to move freely so as to obtain the lowest energy conformers at all stages of the reactions. The exclusion of the remaining enzyme may be regarded as the most severe shortcoming of the present approach. However the thus obtained energy surfaces can still provide valuable insights into the catalytic mechanisms, and be used to discriminate between several alternative pathways. In addition, it has now been shown in numerous examples, that once an appropriate model of the active site is used, the geometric arrangements, kinetic data, and possible reaction mechanisms normally agree strikingly well with X-ray crystallography structures and various experimental data, despite the use of a small model system in gas phase. For each of the systems studied a large number of possible alternative pathways, intermediates, etc, are always investigated. The results shown in the present chapter illustrates the best possible sets of data within the approximations imposed, but should not be viewed as representing 'the full truth'.

3. Galactose Oxidase Galactose Oxidase (GO) from the filamemous wheat-root fungus Fusarium spp. is a mononuclear type 2 copper enzyme that catalyzes the two-electron oxidation of a large number of primary alcohols to their corresponding aldehydes, coupled with the reduction of dioxygen to hydrogen peroxide [1,18]:

RCH2OH

+

02 "') RCHO + H202

The protein is a single polypeptide with molecular mass of ca 68 kDa. To perform the two-electron chemistry, the enzyme utilizes, in addition to the copper center, a protein radical cofactor, which has been assigned to the Tyr272 residue. GO can exist in three distinct oxidation states: the highest state with Cu(II) and tyrosyl radical, the intermediate state with Cu(II) and tyrosine, and the lowest state with Cu(I) and tyrosine. The highest oxidation state is the catalytically active one. The protein radical couples antiferromagnetically with the copper ion, resulting in an EPR silent species. The X-ray crystal structure of the active form of GO has been determined at pH 4.5 and 7.0 [19]. The copper site was found to be close to the surface with essentially square-pyramidal coordination with Tyr495 in axial position and Tyr272, His581, His496 and a water or acetate to be replaced by substrate in equatorial positions (Figure 1). Interestingly, Tyr272 was found to be cross-

150

(~_

Tyr495

His496

Cy

Figure 1. Crystal structure of the active site of galactose oxidase. The substrate is believed to replace the exogenous water in the equatorial position. linked to a cysteine residue (Cys228) through a thioether bond at the orthoposition to the phenol OH. The Tyr-Cys moiety is, moreover, r~-stacked to a tryptophan residue (Trp290), which also controls the entry to the active site. Another interesting feature of the active site is the direct backbone link between the consecutive amino acids Tyr495 in axial position and His496 in equatorial position. The catalytic mechanism proposed for GO is shown in Figure 2 [20]. After the substrate binds to the equatorial copper position (occupied by water or acetate in the crystal structures), the first step is a proton transfer from the alcohol to the axial tyrosinate (Tyr495). Next, a hydrogen atom is transferred from the substrate to the modified tyrosyl radical. This step is known from isotope substitution experiments to be at least partially rate-limiting and probably the major rate-limiting step. The resulting substrate-derived ketyl radical is then oxidized through electron transfer to the copper center yielding Cu(I) and aldehyde product. Based on experiments with various inhibitors, Branchaud and co-workers have suggested that the two latter steps might occur simultaneously in a concerted manner [21]. Finally, Cu(I) and tyrosine are oxidized by molecular oxygen, regenerating Cu(II) and tyrosyl, and giving hydrogen peroxide as product. In accordance with the general guidelines given above for the choice of chemical models, the two histidines were modeled by imidazoles, the equatorial tyrosine was modeled by SH-substituted phenol, whereas the somewhat smaller, but fully adequate, vinyl alcohol served as model for the axial tyrosine. The

151

Tyr495

O \/

~cu"

,.

.,...._

Proton Transfer

His496

\

Step 1 O H

~Cu ~N ~ /

H ~ \

S~ O

HNR

R

H202

Reduction of Dioxygen

02

H

Step 2

I

HydrogenAtom Transfer

3

4 \t

\,H

H Step 3

~N ~ ,

/Cu

H

~ S~

Electron Transfer

~-N/C~ / O

S~

H

H

Figure 2. Proposed reaction mechanism for galactose oxidase. simplest alcohol, methanol, was used as a substrate. The rest of the phenol ring of the axial tyrosine and the backbone link between it and the equatorial histidine (His496) were included as molecular mechanics atoms, using the IMOMM (Integrated Molecular Orbital / Molecular Mechanics) hybrid method [22]. This method uses quantum mechanical and molecular mechanics descriptions for different parts of the system, and it has proven to be successful in the quantification of steric effects in a number of organometallic applications [23]. As in the case of PFL (see below), a charge-neutral model was used for galactose oxidase. This model implies that one of the histidine ligands needs to be deprotonated in order to obtain the correct oxidation state of the copper atom. We start the discussion of the mechanism of galactose oxidase by noting the following. From experimental heats of formation one can calculate that the net reaction catalyzed by the enzyme, with methanol as substrate: CH3OH + 02 ~

CH20 + H202

is exothermic by 10.7 kcal/mol [24] (in our calculations 6.7 kcal/mol). The fact that the reaction is catalyzed by the enzyme does not change this total exothermicity. This sets some restrictions on the energetics involved in the catalyzed reaction. For instance, the proposed electron transfer from the ketyl

152 radical anion to the Cu(II) center cannot be very exothermic, since this would render the oxygen reduction steps rate-limiting, with a barrier higher than the 14 kcal/mol estimated for the H-atom transfer between substrate and Tyr-Cys system. In the calculations, it was found that the first step, the proton transfer from the substrate to Tyr495 (Step 1 in Figure 2), occurs with a very low barrier (less than 3 kcal/mol). The exothermicity was calculated to be 3.2 kcal/mol. One of the important results that come out from the calculations is that the radical site prior to the proton transfer (1 in Figure 2) is not the equatorial cysteine-substituted tyrosine residue, but rather the axial tyrosine (see spin distribution in Figure 3A). The axial position is the weakest one in the square pyramidal coordination of Cu(II), and thus the most natural place for the radical to be in. A series of model calculations with different ligands, ranging from simple OH and water to full phenols and imidazoles, was done, and it was found that all the calculations are consistent in having the radical axially in a Cu(II) complex (data not shown). Stack and co-workers [25] have synthesized model complexes that resemble both the spectroscopic characteristics and the catalytic activity of galactose oxidase. For these complexes, EXAFS and edge XAS experiments indicate that the radical is most likely located axially in the non-square planar coordination of the copper. Calculations by Rothlisberger and Carloni [26] on these model systems confirm this fact. We also recommend the chapter herein by that group, in which the full reaction mechanism of GO has been investigated using Car-Padnello MD methods.

r

S=-.5

c 5

=.11 =-.28

c

~8

19

1.45 ~

A

./-

~

,:s~-20 =.10

S=-.11

B

Figure 3. Optimized structures for galactose oxidase active site before (A) and after (B) the proton transfer from the substrate to the axial tyrosine.

153 In our calculations [2], the radical is after the proton transfer located at the equatorial tyrosine (Figure 3B), inferring that simultaneously with the proton transfer an electron is moved from the equatorial tyrosine to the axial one. Stack's model systems and GO active site behave, hence, very similarly, which shows that it is not the protein matrix that is keeping the radical in the equatorial position. The difference is rather that in the model complexes neither of the two phenolic oxygens is protonated, which makes the optimal radical Site to remain in the weak axial position. The substrate used in the model compounds is alcoholate (-OCH3), which, in contrast to the GO substrate, cannot give a proton to the axial phenol and thereby move the radical site to the equatorial tyrosine. The second step in the proposed mechanism of GO is a hydrogen atom transfer from the substrate to Tyr272 radical (Step 2 in Figure 2). On the basis of isotope substitution experiments, this step has been shown to be at least partially rate-limiting and probably the major rate-limiting step. Figure 4 shows the optimized structure for the transition state of this hydrogen atom transfer. The barrier was calculated to 13.6 kcal/mol. It is known that turnover rates of alcohols exhibit strong substituent effect [27]. For instance, galactose has a turnover rate of 800 s-~, while for ethanol it is only 0.02 s-~. Assuming the hydrogen atom to be fully rate-determining, a barrier of ca 14 kcal/mol can be estimated using the kinetic data for galactose as substrate [28]. For ethanol, the

1.37

s=-.31(

s=-.07

Figure 4. IMOMM(B3LYP/MM3) optimized geometry of the transition state for the proposed rate-limiting hydrogen atom transfer.

154

barrier can be estimated to be ca 6 kcal/mol higher than for galactose. Although the DFT-calculated barrier (for methanol) is somewhat lower than the experimental estimation, it does indeed provide strong support for the proposed mechanism. As seen in Figure 4,' the critical C-H bond has stretched to 1.36/k at the transition state, and the H-O bond about to form is 1.24 A. At the transition state, one spin is located at the copper (S=0.49) and the other is shared by both the tyrosine and the substrate (S=0.15 and S=0.49, respectively). The hydrogen atom carries 0.07 of the unpaired spin. Consistently with the tyrosyl radical being a n-radical, we note that the hydrogen atom is transferred perpendicularly to the phenol ring plane. Stretching the phenol O-H bond in the plane of the ring would instead lead to a high-energy t~-radical. The hydrogen atom transfer is proposed to result in a substrate derived ketyl radical (3), which then would be oxidized through electron transfer to the copper center, yielding Cu(I) and the aldehyde product (4). As mentioned above, these two steps have been proposed by Branchaud and co-workers to occur in a concerted manner [21 ].

1.3" S=-.52

Figure 5. IMOMM(B3LYP/MM3) optimized structure of the ketyl radical intermediate (structure 3 in Figure 2).

155

By moving from the transition state structure towards the product, we were able to localize the proposed radical intermediate in the IMOMM calculation (Figure 5). The energy of the intermediate is 4.9 kcal/mol down from the transition state, making the hydrogen atom transfer step endothermic by 8.7 kcal/mol. Without including the MM part, all attempts to localize the ketyl radical intermediate failed, indicating that this intermediate is very unstable with the barrier for its collapse to the closed shell Cu(I) and aldehyde product being very small. In practice, this radical intermediate is therefore probably impossible to detect. The ketyl radical intermediate (3) is hence unstable and will readily reduce the copper center, yielding Cu(I) and aldehyde (4). The electron transfer (ET) step was estimated to be exothermic by 36 kcal/mol, by Wachter and Branchaud [_28]. Due to the smallness of the model employed, the closed shell Cu(I) + aldehyde system (4) will, when optimized, have a largely distorted geometry. Although the backbone link between Tyr495 and His496 included in the IMOMM calculations reduces the distortion somewhat, it is clear that this species is overstabilized in the model calculations. It can only serve as an upper limit to the amount of energy that can be gained. The exothermicity is calculated to around be 14 kcal/mol relative to the ketyl radical intermediate. The protein matrix will of course prevent such a large distortion and we estimate the energy of the complex, with proper coordination before the distortion to be around 5 kcal/mol loweY than the energy of the ketyl radical intermediate. This estimation is based on the energy of the system in the first few steps of the geometry optimization, i.e. before the distortion becomes very large. Energy is instead gained through the binding and one-electron reduction of dioxygen. Assuming the aldehyde product is released at this stage, and that dioxygen occupies its coordination position, 02- is found to bind to copper by 20.6 kcal/mol more than the substrate alcoholate (-OCH3). The structure of this complex is shown in Figure 6. This energy is reasonable, because, as discussed above, large exothermicity in this step would render the reduction of 02 ratelimiting. The calculated potential energy curve for the steps discussed above is displayed in Figure 7. The role of the tyrosine-cysteine cross-link is of fundamental mechanistic interest. It has been suggested that this thioether bond is in part responsible for the 0.5-0.6 V lowering of the oxidation potential of this species compared to normal tyrosine [29]. The C228G mutant has, furthermore, been shown to have 10,000 times lower activity than the wild-type enzyme, and also migrates slower on gel electrophoresis [30]. The crystal structure of the mutant did not show much change in the main chain or the copper binding site due to the mutation, except, of course, for the missing thioether bond. It was proposed that

156 destabilization of the tyrosyl radical due to less electron delocalization caused this activity decrease.

,) N) .... .

S=.55

t

s1.38

OQ~

....

S=-.55

Figure 6. IMOMM(B3LYP/MM3) Optimized structure of 02- bound to GO active site. There are, however, many pieces of evidence that the cysteine link only causes small perturbations in the electronic structure and energetics of tyrosine. Electrochemical experiments by Whittaker et al [31 ] showed that the pKa of omethylthiocresol was only 0.7 pH units lower than for cresol (9.5 vs. 10.2). Babcock and co-workers have shown, based on EPR and ENDOR experiments on both apo-enzyme and model alkylthio-substituted phenoxyl radicals, that the sulfur cross-link only induces small perturbation in the spin distribution of the tyrosyl radical [32]. No big shift in the g-tensors between unsubstituted and methylthio-substimted radicals was observed. Since this kind of shift is expected when heavy elements carry some of the spin in organic radicals, the conclusion was that the sulfur center possesses only a small part of the unpaired spin. We have conducted ab initio multiconfigurational linear response g-value calculations of unsubstituted and sulfur-substituted phenoxyl radicals and shown that the shift in g-tensor is as small as 0.0008 in the gxx-Component (2.0087 vs. 2.0079 in t). The other components were virtually unchanged, thus confirming the experimental results [33]. By means of density functional calculations, it was also shown that the thioether bond has very small effects on the hyperfine couplings and spin distributions [34]. The odd-akernant spin pattern of the tyrosyl radical was

157 essentially unbroken, with the sulfur center only having ca 0.12 of the unpaired spin. The full spin density distributions, calculated with several different density functionals, are displayed in Table 3. We have also calculated the sulfur substituent effect on the O-H bond dissociation energy (BDE) of phenol [35]. The BDE of the sulfur-substituted phenol was found to be only 1.7 kcal/mol lower than the unsubstituted species. As for the effects of the cysteine cross-link on the catalytic mechanism, the calculations were re-done without including the sulfur linkage. Both the energetics and the geometrical structures were found to be almost identical. For example, the barrier for the critical hydrogen atom transfer step was calculated to be about 1 kcal/mol higher with the sulfur moiety included, i.e. the sulfur link actually makes this step proceed somewhat slower, but this result of course falls within the error margin of the methods used. Evidently, the thioether bond has very small electronic effect on the tyrosine. The role of the cross-link could be o:f structural nature, keeping things in place. Table 3. Mulliken spin population distributions for unsubstituted (Tyrosyl) and ethykhio-substituted (Tyrosyl-S) tyrosyl radicals calculated with various DFTfunctionals.

H

H

H

N

H

~ s

H

o

atom

C1 C2 C3 C4 C5 C6 O S

o

Tyrosyl B3LYP BLYP B3P86 PWP86 0.40 -0.16 0.31 -0.11 0.31 -0.16 0.43 .

0.35 -0.10 0.26 -0.05 0.26 -0.10 0.39 . .

H

0.41 -0.17 0.32 -0.11 0.32 -0.17 0.42 .

0.36 -0.10 0.26 -0.03 0.25 -0.09 0.37

Tyrosyl-S B3LYP BLYP BLYP PWP86 0.34 -0.16 0.28 -0.06 0.23 -0.11 0.35 0.11

0.28 -0.10 0.23 -0.00 0.18 -0.05 0.31 0.14

0.35 -0.17 0.28 -0.05 0.24 -0.11 0.35 0.12

0.28 -0.09 0.21 0.02 0.18 -0.03 0.28 0.15

To summarize this section, the theoretical calculations [2] strongly support the mechanism proposed for galactose oxidase. It was shown that the proton transfer step proposed to initiate the oxidation of the substrate is very fast and just slightly exothermic. The rate-limiting hydrogen atom transfer step has a calculated barrier of feasible 13.6 kcal/mol. The proposed short-lived ketyl radical intermediate has been localized, and it was argued that the subsequent

158

electron transfer fxom this to Cu(II) cannot be very exothermic. High exothermicity at that point would render the reduction of 02 rate-limiting. The radical site prior to the initiating proton transfer is, moreover, proposed to be located at the axial tyrosine (Tyr495), rather than at the equatorial thioether substituted Tyr272, as previously suggested. This would bring consistence between GO and model experiments by Stack, where there are strong indications that the radical resides axially. The cysteine cross-link, finally, was shown to have a very small effect on energetics and spin properties of the system.

hydrogen atom transfer 12

-

10.4 e--transfer 5.5

4 0

E

0

o --~

-4

. <3 .0.0/~ 1

!

2

-3.2

),, -8

proton transfer

t..-

w > -.~

rr

3

-10.7

-12 .

4

-16 .

-14 ~Oe-bindin

\

~ J

-20

O2-reduction

-24 -23.8 -28 Reaction Coordinate

Figure 7. Calculated, IMOMM(B3LYP/MM3), potential energy surface for the mechanism of galactose oxidase.

4. Pyruvate F o r m a t e - L y a s e

Pyruvate formate-lyase (PFL) catalyzes the reversible conversion of pyruvate and CoA into acetyl-CoA and formate [36,37]" O -O20-'~0H3

O d-

CoA--SH

-

-

CoA_S-'~CH3

+

HCO2

159 The enzyme is a homodimer composed of 85 kDa monomers and is essential for the anaerobic glucose metabolism in Escherichia coli and other bacteria. PFL exhibits tWO-Step ping-pong kinetics with acetylated enzyme intermediate. The catalytic power is high, with kcat = 770 s-~ for the forward direction and k c a t "- 260 s-1 for the backward direction. The active enzyme contains a stable organic radical, which has been assigned to the Gly734 residue [15]. This was the first example of a radical enzyme with the radical located at the protein mainchain. Glycyl radical has also been found in anaerobic ribonucleotide reductase [16,38]. The stability of the glycyl radical is usually explained by means of the so-called capto-dative effect [37]. This occurs when the radical center is located between an electron donor (the amino group) and an electron acceptor (the amide carbonyl). The combined effect of these two groups gives an enhanced radical resonance stability. In the calculations [3], protein-bound glycine is modeled by adding the p_eptide bond on each side of the glycine, CHO-NH-CHz-CO-NH2 (see Figure 8).

S=.ll

g

, S

5

~ 1.22 S=.ll Figure 8. Optimized geometries of the model used for protein-bound glycine and its C~-radical. Mulliken spin populations are also shown.

As seen from the figure, the spin is delocalized also to the backbone of the adjacent amino acids. The spin populations on the carbonyl oxygen and the nitrogen next to the radical carbon (0.11 and 0.06, respectively) confirm clearly the capto-dative hypothesis. However, there is spin also further out in the backbone chain: the oxygen and the carbon of next carbonyl has 0.11 and 0.04, respectively, and the nitrogen on the opposite side has 0.05. From these spin populations and also the bond distances displayed in Figure 8, additional resonance structures can be drawn for protein-bound glycyl. These are displayed in Figure 9. An extended model of glycine is hence important to in order to account for the resonances in the protein-bound radical. This effect was also seen in Table 2, illustrating the dependence of the radical HFCC' s on the system size.

160

O

H

H

O

H

H

N\R H

N\R

O

H

A

"O

H

0 D

R.~

O*

H ~+ H

B

H

H

O

O

H

0 E

N\ R O C

H

H

H

O

H

H

H

0 F

Figure 9. Resonance structures present in protein-bound glycyl radical. A-C represent the so-called capto-dative effect, whereas D-F are resonances due to the backbone of neighbouring amino acids.

It has been established, by means of site-directed mutagenesis experiments, that three amino acid residues are essential for the overall catalysis, namely the glycyl radical (Gly734) and two consecutive cysteines at positions 418 and 419. Two mechanisms were proposed for PFL at the time when the theoretical study was performed. Although based on the same experimental information, these two mechanisms are quite different (Figures 10 and 11). In the mechanism proposed by Knappe and co-workers [36] (Figure 10), the protein radical is transferred from glycyl to Cys418 after pyruvate has added to the Cys419 building a thiohemiketal moiety. The thiyl radical of Cys418 then forms an adduct to the carboxyl of the thiohemiketal. Next step is an intramolecular hydrogen atom transfer, yielding alkoxy radical intermediate which then undergoes the homolytic C-C bond cleavage. Another intramolecular hydrogen atom transfer occurs in the Cys418-formate radical adduct, resulting in an oxy radical that dissociates to form free formate and Cys418 radical. The thiyl radical at Cys418 is, finally, quenched by Gly734, completing the first half reaction. The subsequent transfer of the acetyl group from Cys419 to CoA is proposed to use Cys418 as a nucleophilic relay. The second mechanism is due to Kozarich and co-workers [39] (Figure 11). The initial step here is the abstraction of a hydrogen atom from Cys419 by the glycyl radical, forming a transient thiyl radical. Addition of this thiyl radical to the keto group of the pyruvate results in the formation of a tetrahedral oxyradical intermediate. This intermediate collapses into an acetylated cysteine and

161

Gly"

Gly(H) 4

Cys41

o II

/ t ~ ' s - ~ OH / co~

......

C y s 4 1 8 ~ H H3c/C~co2-

CH3

~oO~

~o-

~

~

--,.

o

CH3 o

OH

1L

/~

~

-~ ~:-~

Giy- Gly(H)

~~o OH

HCO2-

Figure 10. The reaction mechanism proposed by Knappe and co-workers [36]. a formyl radical, which is then reduced to formate by hydrogen atom abstraction from the glycine residue, regenerating hence the stable glycyl radical. A step of transesterification between C419 and C418 takes place before the reaction is completed by the CoA-dependent thioester exchange.

H2 I~ Cys419 ~-~SH C~41~

~

|

H3C.,,~3~COa.

02" ~

"-

sH 0

c

"COs SH

~

3

~

s

CH3 HCO2"

Figure 11. The reaction mechanism proposed by Kozarich and co-workers [39].

162

As discussed above, in the calculations, a large model of the glycyl residue (CHO-NH-CH2-CO-NH2) proved needed, in order to correctly account for the resonances in the protein-bound glycyl. Cysteine was modeled by methylthiol, HSCH3, according to the discussion in the Methodology section above. Also in this study, we chose to work with a charge neutral model, i.e. the total charge of the species considered Was chosen to be zero. Pyruvate was accordingly modeled by pyruvic acid, and formate by formic acid. The first step of the catalytic cycle proposed by Kozarich is the creation of a transient thiyl radical at the Cys419 position. Assuming that the cysteines are in close spatial proximity to the glycyl radical, the barrier for the direct hydrogen transfer reaction between glycyl and cysteine was calculated to 9.9 kcal/mol. The Ca-H bond strength of the glycine model used is 79.3 kcal/mol and the S-H bond strength of the cysteine model is 81.9 kcal/mol. This makes the hydrogen atom transfer from cysteine to glycyl endothermic by 3.4 kcal/mol. The optimized structure for the transition state is displayed in Figure 12. The spin at the transition state is distributed mainly on the sulfur and the glycyl Ca centers (0.42 and 0.43, respectively). However, also the carbonyl oxygens have some spin, 0.05 - 0.07. S=.05 1.55 1.42

S=.42 1.84

S=.07

S=.07

Figure 12. Optimized transition state structure for the direct hydrogen atom transfer from cysteine to glycyl radical. The next step is the addition of the thiyl radical to the carbonyl carbon of pyruvate, yielding a tetrahedral oxy-radical intermediate. The calculated energy of this intermediate, relative to the free reactants, is +9.9 kcal/mol, and the barrier for its formation is calculated to 12.3 kcal/mol. The barrier for the dissociation of the radical intermediate into acetylated cysteine and formyl radical is calculated to be only 2.8 kcal/mol with an exothermicity of 3.9 kcal/mol. Taken together, the total reaction: Cys419o + pyruvate

Cys419-acetyl + formylo

163 is hence endothermic by 6.0 kcal/mol. These results show that the scenario proposed, involving the tetrahedral radical intermediate, is indeed energetically plausible. The structures and spin distributions of the two transition states (TS1 and TS2) and the tetrahedral intermediate are shown in Figure 13.

~ S=.15 1.52 ~,~ 1.66 ~1.19

1.52

S = . 2 ~

S=.49 .

A

4---

B

4

F

1.8Z f

S=-.04

.... ~

~

~S=.21

1 S=.31 ~""~" S=.06

C

Figure 13. Formation and collapse of the tetrahedral oxy-radical intermediate. Optimized structures for A) transition state of the thiyl radical addition to pyruvate (TS 1), B) tetrahedral oxy-radical intermediate, and C) transition state of the dissociation of formyl radical (TS2). The energies of these species are 12.3 kcal/mol, 9.9 kcal/mol, and 12.7 kcal/mol, respectively, relative the energy of (methylthiyl + pyruvic acid). The spin at TS1 is distributed on the sulfur atom (0.52), the carbonyl oxygen (0.24), and the carboxylic CO group (0.21). The S-C2 distance is 2.13 and the C2-Ccarboxyl bond is elongated to 1.66 /k (1.55 /k in pyruvate). The tetrahedral radical intermediate exhibits a somewhat different spin pattern. The spin is mainly concentrated on two centers, the sulfur atom (0.49) and the carbonyl oxygen (0.55). The carbonyl C2-O bond length is clearly of single bond nature (1.34 A). The S-C2 distance is 1.92 A and the C2-Ccarboxylbond is 1.56 A. At the second transition state (TS2), the spin density is moving over to the carboxylate group (0.58), although some of the radical character still remains on the sulfur (0.23) and carbonyl oxygen (0.20). The critical C2-Ccarboxyl bond is 1.98/k and the S-C2 distance is 1.88/k. Heterolytic addition of thiol to pyruvate was also considered. It was found that the direct thiol attack at the carbonyl carbon of pyruvate, yielding a nonradical tetrahedral intermediate has very high barrier (37.6 kcal/mol), although the intermediate has plausible energy; 1.6 kcal/mol over the free reactants (optimized structures are given in Figure 14). The possibility of a direct attack is thus ruled out. A much more plausible barrier is found when letting a carboxyl group to mediate in this reaction. The barrier dramatically drops to 12.0 kcal/mol (structure shown in Figure 14). This is clearly

164 competitive with the radical reaction, provided a carboxylate group-containing residue (Glu or Asp) is present to do the catalysis. No such group is, however, known to participate in the catalytic reaction of PFL.

1.42

A

B

C

Figure 14. Optimized structures for A) non-radical tetrahedral intermediate formed upon addition of cysteine to pyruvate, B) transition state for direct thiol attack on the carbonyl of pyruvate, and C) thiol attack mediated by a carboxyl group. The localized tetrahedral intermediate of the radical pathway will readily dissociate and release formyl radical. This reactive radical is proposed by Kozarich et al to abstract a hydrogen atom from Gly734, hence regenerating the stable enzyme radical at that site. The calculations show that this proposal is perfectly feasible, having a barrier of 4.9 kcal/mol and an exothermicity of 17.5 kcal/mol. However, bearing in mind that the subsequent step is an acetyl group transfer between the cysteines, we proposed that the formyl radical may instead abstract a hydrogen from Cys418 rather than from Gly734. The barrier for this is very low (1.1 kcal/mol), although the exothermicity (14.1 kcal/mol) is slightly lower than for the reaction with glycine described above. The thiyl radical thus created at Cys418 will now allow for a radical mechanism for the transfer of acetyl from Cys419 to Cys418. This transfer was originally proposed by Kozarich et al for two reasons. In acetylated PFL (the result of addition of pyruvate to activated PFL, in the absence of CoA), glycyl can still exchange its hydrogen, indicating that Cys418 is the site of acetylation, since Cys419 is known to be required for that exchange. From site-directed mutagenesis experiments it is also known that Cys418 is the primary residue participating in the thioester exchange with CoA, since thioester exchange to

165

CoA is observed for C419S mutant, but not for C418S. Note, however, that both the suggested mechanisms are consistent with the experimental observations. To move the acetyl group homolytically between the cysteines has a computed barrier of quite reasonable 11.6 kcal/mol. In Figure 15, the transition state structure for this reaction is presented. This thermoneutral occurs without the intermediacy of tetrahedral oxy radical intermediate.

S=.52 1.51~ ~ = - .04 222"

Figure 15. Optimized structure and spin population distribution of the transition state of the homolytic acetyl transfer between cysteines. A direct nucleophilic attack by the sulfur of non-radical cysteine on the carbonyl carbon of acetylated cysteine, yielding a tetrahedral intermediate, was also considered. As in the case of nucleophilic attack of cysteine on pyruvate, the intermediate lies relatively low in energy (+9.0 kcal/mol), but barrier is very high (41.2 kcal/mol). For the transfer of the acetyl from Cys418 to CoA we propose a similar homolytic radical mechanism as for the acetyl transfer between the two cysteines. For computational point of view, these two steps are identical, because the same model (methylthiol) was used for both cysteine and CoA. We propose, therefore, the following steps for the acetylation of CoA: .

2.

CoA-SH + Cys419- ~ CoA-So + Cys419 CoA-So + Cys418-acetyl ~ CoA-acetyl + Cys418.

The first step (1) is a simple hydrogen atom transfer, where the Cys419 radical abstracts a hydrogen from CoA-SH. This thermoneutral step has a calculated barrier of 2.4 kcal/mol. Step (2) is identical to the acetyl group transfer step between the cysteine residues (Figure 15) with a barrier of 11.6 kcal/mol. The enzyme can now either take another substrate, or regenerate the glycyl radical. The latter possibility could be accomplished through direct hydrogen atom transfer from Gly734 to Cys418 (provided proximity between

166 them), or using C419 as radical relay. These final steps are all calculated in previous steps and have low barriers. The full mechanism based on the calculations is summarized in Figure 16.

~ '-

Cys418~SH

u

9CO2-

Hac~C~coz.

~SH

N~SH

N~SH

oO~

g

~176176 CH2

/~CH2

H

~

CoA-S~

(..SH

O CoA-S~

~

CH2 0

/~CH2 CoA-SH

CHa

Figure 16. New reaction mechanism proposed for PFL [3]. During the preparation of this review, two papers were published describing the X-ray crystal structure of PFL. The first structure was solved by Goldman and co-workers but is lacking 125 C-terminal residues, including the essential Gly734 [40]. The second structure is complete and is due to Kabsch and co-workers [41]. Gly734 and Cys419 were found to be very close to each other, only 3.7 /k, confirming biochemical data and justifying the theoretical models. The other active site cysteine residue (Cys418) was found to be more buffed inside the protein, but in close proximity to Cys419, allowing for hydrogen transfer between these two residues. Kabsch and co-workers also crystallized PFL in complex with the substrate analogue oxamate, which differs from pyruvate in having an amino group instead of the methyl (Figure 17). This structure shows that Cys418 is perfectly located to attack the C-2 carbon of the substrate. Two arginine groups are, furthermore, thought to bind and stabilize the substrate. The structural results have some implications on the catalytic mechanism. The position of the substrate suggests that Cys418, and not Cys419, performs the radical attack on pyruvate. This would require two hydrogen atom transfers, first from Cys419 to the glycyl radical, and then from Cys418 to Cys419. This is reminiscent of the long-range hydrogen atom transfer in ribonucleotide reductase. There, the radical is transferred some 35 A from the tyrosine at the diiron site in R2 to the active site cysteine in R1 (see below). The function of Cys419 is, hence, just to mediate the radical transfer between

167 Gly734 and Cys418. This renders the acetyl transfer between the two cysteines unnecessary, because the acetyl is already at Cys418. Since the two cysteines were modeled in exactly the same way in the calculations presented above, all the results found for the addition of Cys419 radical to pyruvate also apply to Cys418.

G

\ ('~

- Cys418

oxam~

Arg176

Figure17. X-ray structure of the active site of PFL in complex with the substrate analogue oxamate. The glycyl radicals found in PFL and anaerobic RNR are remarkably stable. However, exposure of these anaerobic enzymes to oxygenated solutions are known to result in cleavage of the peptide backbone at the site of the glycyl radical [15,42]. Recently, Reddy et al reported on a detailed experimental investigation of the oxidative degradation of wild-type PFL, and samples where either or both of the two cysteines essential for catalysis were substituted by alanine [43]. Using mass spectrometry and EPR spectroscopy they were able to observe the well established products resulting from fragmentation at the Cc~-N bond of the glycyl radical [15], as well as products indicative of cleavage at the C1-Ccz bond. In addition, EPR data suggested the existence of a long-rived sulfinyl radical (R-SO-) at C419 in the wild-type and C418A mutant system, and a peroxyl radical in the C419A and C418AC419A mutants. Based on these observations, three alternative reaction mechanisms were suggested. All these, plus some additional alternatives, have recently been studied theoretically [4]. Based on a large set of reaction pathways, the most plausible alternative (a somewhat modified form of the main mechanism proposed by

168

Reddy et al) can be summarized as follows (Figure 18). Initially, 02 will add to the glycyl radical center in a barrier-free reaction (AE = -7.2 kcal/mol). The glycyl-peroxyl radical will then abstract the thiol hydrogen from C419 in an almost thermoneutral reaction with a barrier of 10.4 kcal/mol, followed by OH transfer from Gly-OOH to Cys-S-. This reaction has a similar barrier to that above, but is exothermic by ca 30 kcal/mol. The overall exothermicity from the initial starting point with G734~ 02 and C419, is 34.7 kcal/mol. The glycyl-alkoxy radical (G734-O-) can now easily abstract the C419SOH hydrogen to form the observed metastable sulfinyl radical and t~-hydroxyglycine. The hydroxy-glycine readily undergoes hydrolysis, and gives the observed, 'normal', fragmentation products resulting from cleavage of the Ct~-N bond. The H-abstraction step is essentially barrierless, and again exothermic by ca 30 kcal/mol. Relative Energy kcai/mol 20

TS

TS

o:o Gly* -20 - +02 +RSH

-40

-7.2 . Gly-OO +RSH

-4.7 TS

Gly-OOH +RS*

-32.6

-34.7

-35.5

Giy-O* +RSOH

-49.2

-60

-66.2

H2NCHO + OC*OH Gly-OH +RSO*

Figure 18. Main reaction pathways of oxidative degradation of PFL.

Alternatively, the glycyl-alkoxy radical may also undergo C-C bond cleavage. This reaction has a barrier of only 2 kcal/mol, and is exothermic by ca 14 kcal/mol, and will hence explain the observation of Ctx-C1 fragmentation products in the mass spectra. As noted above, very recent Xray-data show that C419 and G734 are in close contact at the active site, whereas C418 and G734 are further apart. This will explain the existence of stable peroxy radicals (Gly-

169 0 0 - ) in the C419A mutants, whereas such species are believed to be transiently observed in normal C419 type enzymes. As mentioned above, several optional reaction pathways were also investigated in this study [4], but none of these was able to explain all experimental observations, or were energetically feasible.

5. Ribonucleotide Reductase

Ribonucleotide reductases (RNR) constitute a large group of essential enzymes with a diverse array of primary as well as quaternary structures. Common for the enzymes is that they catalyze the rate-determining step in DNA biosynthesis, the reduction of ribonucleotides into deoxy-ribonucleotides (Figure 19) [44,45]. (P)PPO

Base

H~4* O1 ' ~ H H----~3'

/

H.-O

(P)PPO,~

RNR

O

Base

H

H

2' L.-H

\

O~H

ribonucleotide

H.-O

.

deoxyribonucleotide

F i g u r e 19. Net reaction of Ribonucleotide reductases

The RNRs are divided into four classes, depending on the cofactors utilized to catalyze the reaction [45]. Class I RNR, which is found in e.g. mammals and E.coli bacteria, employs a stable neutral tyrosyl radical coupled to a di-iron (Fe:O2) cluster [46]. Class II uses 5'-deoxy-5'-adenozyl-cobalamin [47] (the active form of vitamin B12 - see also the chapter by Smith, Wetmore and Radom). Class III is also found in E. coli, when grown under anaerobic conditions, and uses a neutral glycyl radical as cofactor, similarly to the previously described anaerobic PFL enzyme [48]. Class IV, finally contains what is again believed to be a tyrosyl radical, this time linked to a di-manganese cluster [49]. In addition, class I RNRs have been divided into subclasses la and lb, differing in e.g. their expression mechanism [50]. We will in this chapter consider aerobic Class I RNRs only. The systems have been the subject of extensive experimental work, including EPR spectroscopy, isotopic labelling, inhibitor mechanisms, mutagenesis and kinetics studies, and a large number of excellent reviews are available summarizing the present knowledge ([1] and references therein). Besides the actual catalytic machinery, large efforts have also been devoted to understanding the activation processes, i.e., the formation of the di-iron complex

170 and the generation of the stable tyrosyl radical. These latter aspects will not be considered here; instead the reader is, e.g., referred to the recent review by Stubbe and van der Donk [1 ].

Cys462 Cys225 [substrate]

Trp,

Cys439

R1

'.

Tyr730 '. Tyr731

,

35 A

\

H.

S = 0.33

" "O

'

Trp48

(Tyr356) .' .. .~Asp237

Aspz~7

." S = 0 . 0 0

R2

"" V " ~ \~ Tyr122" ... Glu115 W Asp84

H /

Ir~NN

S = 0.12

His241

H.._

His11'8 Glu238 W. ~Fe/O~Fe ~ His241

A

OI ~ll i

Trp111

I/H20

H"uE;.Fe~OH (Tyr1220*i ..

~/O S = 4.00 Asp~

B

Figure 20. A. Model of interaction between tyrosyl radical (Tyr122) at R2 subunit and active site residue Cys 439 at R1 subunit of class I RNR. W indicates ligated water molecules. B. Computational model of the R2 sub-system, used for modelling the initial stages of the radical transfer. The class I RNRs were discovered in the early 1950's by Reichart et al [46], and was the first enzymatic system that could be shown unequivocally to harbour a stable amino acid radical, by means of EPR spectroscopy [51]. The system is known to contain two loosely connected homodimers, with two R1 or two R2 subunits. The enzyme requires that the two homodimers are connected in order to function. The tyrosyl radical is located in one of the R2 subunits and is connected to the active site at the R1 subunit via a 35 /k long chain of hydrogen bonded amino acids (Figure 20). Substituting any of the amino acids along the pathway by a residue less prone to H-bonding and H-atom migration results in inactivation or significantly reduced turnover rates of the enzyme.

171 The R1 active site harbours five conserved residues, Cys225, Cys439, Cys462, Glu441 and Asn437. Experimental evidence suggests that as the substrate enters the active site pocket, the radical site is triggered to migrate from Tyr122 up to one of the cysteine residues (Cys439) at the active site. Once the radical character has entered the active site, the substrate is able to undergo radical catalyzed conversion from ribose to deoxyribose, including loss of water and formation of a disulfide bridge between residues C225 and C462, that hence serve as reducing equivalents for the nucleotide reduction. Upon completion of the catalysis, the thiol radical is regenerated at C439, and the radical character is transferred back to the Y122 residue buffed deep in the R2 subunit. Figure 21 displays the catalytic mechanism proposed by Stubbe, based on a large compilation of experimental data [1,44,52].

PPEL. 439

Base H

PPQ~

H

439

s,

Step 1

H

O"H

O

O

I

I

439

H

SH

-

H-O

PPO...

Base

E,,

/

\

? / H

e

Step

2

Base H

H

SH

O"H I-t20

H

.I

o E ~ / ~ L" OH

I

S-

SH

I

I

C462/C225

I

3

Step

PPO~ 4,-]9

Base H

PPEL.

H

439

S

Step 5 9 0

H

PPO~

H

439 Step 4

SH

~

HO

Base

/

s

s

H

//

\H

HO

,/~o-

H

SH

\

0

0

/3~.. o-

Base

H

O

$

s

E,d

OH

r

,~

I

S

I

Figure 21. RNR class I catalytic scheme as proposed by Stubbe (exp.) [ 1]. In order to understand the radical transfer mechanism between Tyr122 and the active site, we first need to consider the protonation state of the tyrosyl radical. In Table 4 we list the EPR parameters ((~-protons) and unpaired spin density distributions of neutral vs charged tyrosyl radicals, and compare the data with results obtained for Y122 in wild type E. coli RNR. From the data listed, it is clear that the tyrosyl radical is neutral, which has important implications for the radical transfer- i.e., that this is not a case of pure electron transfer but

172 rather H-atom or alternatively coupled electron-proton transfer. This also agrees with theoretical studies implying that pure electron transfer between Y122 and C439 would be endothermic by as much as 40 kcal/mol, and hence most unlikely [53]. Table 4. Carbon and oxygen spin densities, and o~-proton HFCCs of neutral ethyl phenoxyl radical and ethyl phenoxyl radical cation. System E. Coli RNR Tyr122*

[541

CH3CH2C6H40*

CH3CH2C6H4OI-I+*

Center C1 C2,C6 C3,C5 C4 O C1 C2,C6 C3,C5 C4 O C1 C2,C6 C3,C5 C4 O

Spin 0.38 -0.08 0.25 -0.05 0.29 0.39 -0.12 0.28 -0.03 0.37 0.43 -0.05 0.16 0.21 0.20

aH Axx o~HAyr

-9.6

1.7 -7.0

o~HAzz 2.7 -2.8

0.5

1.6

2.4

-8.8

-6.6

-2.0

-0.9 -6.0

-0.5 -4.7

1.7 -0.5

The model employed to mimic the H-atom transfer pathway can be divided into two parts - the first including the Tyr - Fe - His - Asp - Trp system of the R2 subunit, and the second including two neigbouring tyrosines Y731 and Y730 and the final C439 residue of the R1 subunit (see Fig. 20). The connectivity between the R1 and R2 subunits is not fully established as yet, due to difficulties in isolating and crystallizing the complete enzyme without it falling apart. The most likely route is via Trp48 to Tyr356, which is located close to the surface of R2, and from there onwards via Tyr730 in the R1 unit. The initial stage involves radical transfer from water ligated to the Fe(III,III) cluster over to Y122, as displayed in Figure 20. Initially, all spin (4.00) is located on the Fe(III) iron included in the model. Upon H-atom transfer from water to tyrosyl a spin of 4.04 is found on Fe(III) (Figure 22), indicating that we preserve the Fe(III,HI) cluster, rather than passing via the mixed valence Fe(IV,III) cluster. Also notable is that there is an immediate build-up of charge and spin on the Trp48 residue (spin goes from 0.33 to 1.07, charge increases from +0.20 to +0.73), whereas the charge on the water/OH ligand that has donated its hydrogen to Y122 goes from +0.16 to -0.36. This is indicative of electron transfer between W48 and water caused by the initial H-atom transfer process. The situation is hence very similar to that seen in cytochrome C peroxidase, in which electron transfer is seen from a Trp residue, via an Asp-His

173 sequence, to a heme-bonded iron upon cleavage of 02. The formed Trp radical cation hence retains its proton, albeit it in the equilibrium structure has moved closer to the hydrogen bonding oxygen of Asp237.

(*OTyr356) H \

S = 1.07

O i i

H... o /jj~/AsP237 ."

H.

S = 0.00

" "O

.'

s = 0.0o

,,~AsP237 S

=

0.00

o

N/

H

His241~/[~ N/) H._ (Tyr,22OH).-

.,~N) S = 0.11

AsPs4 A

S = 0.09

His241

I/H20 ~O S = 4.04

,"

H.

I/H20 %F%o.

(TYq22OH)'"

~,~"' S = 4.02 AsPa4

B

Figure 22. A. Optimized intermediate in R2 radical transfer chain, after H-transfer to Tyr122. Spin is immediately localized to Trp48. B. Final step in R2 subunit, obtained after hydrogenation of Asp237 (radical transfer onwards to R1 subunit) [5]. The energetics of these initial stages show an essentially thermoneutral process, i.e., the energy of formation of the Trp radical cation and rupture of the O-H bond of the ligated water is almost identical to the O-H bond strength of tyrosine, 86.5 kcal/mol. Dielectric effects are shown to beimportant in this step, due to the large charge transfer, and leads to an increased O-H bond strength of the ligated water by as much as 9.6 kcal/mol, thereby bringing it closer to the OH bond strength of tyrosine. Once the Trp48 radical cation is formed, the radical character should be transferred on via Tyr356 to the R1 unit. This is modelled by hydrogenating Asp237 (since Trp48 is still protonated), Figure 22B, and again the binding energy is almost identical to that of tyrosine. The entire radical transfer within the R2 subunit is hence essentially thermoneutral.

174

3.251

~,O

1.18e

I H.

1.215 O Figure 23. Transition state structure for H-atom transfer between Y731 and Y730 (bond lengths in ~gstr6m). Note that the two phenolic rings are ~-stacked. The second step involves radical transfer within the R1 unit, and could be shown to involve pure H-atom transfer [5]. In these calculations, the protein backbone joining Y730 and Y731 had a pronounced effect in that it keeps the residues in a geometry resembling that of the transition state (Figure 23). The barrier for H-atom transfer - is only 4.9 kcal/mol, and the dielectric effects negligible. Allowing the tyrosines to move freely (i.e. removing the backbone) instead raises the barrier to 9.5 kcal/mol due to the more stable structure of the reactants/products. The TS looks highly similar to that observed in the full calculations. The H-atom transfer nature of this step (rather than electron + proton transfer) is clearly manifested in the geometry, in that the H-atom bends ca 50 ~ out of the plane of the tings, and that it throughout retains its electron.

Relative

Energy

10 - kcal/mol

TS

m

_

TS

Cys-SH / - Tyr-OH / Tyr-O* / 0.0

+4.9

+8.1

Cys-SH

Tyr-O* Tyr-OH 0.0

Cys-S* Tyr-OH Tyr-OH +0.4

Figure 24. Energetics for R1 H-atom transfer steps (Y730-Y731-C439). All energies are ZPE corrected and include dielectric effects. The final step of the radical transfer is H-atom migration between tyrosyl radical Y730" and C439. This has a barrier of ca 8 kcal/mol, and is only slightly endothermic (0.4 kcal/mol). The overall radical transfer between Y122(R2) and

175

C439(R1) is hence characterised by thermoneutrality and very low barriers requisites for a fast "radical shuttle" up to the active site when needed, as well as back into the protected environment once the substrate reaction is over (Figure 24). The substrate mechanism has been investigated theoretically in detail by Siegbahn [6], based on the mechanism previously suggested by Stubbe (Fig. 25). Until recently, no intermediates had been isolated within the catalytic cycle, and the proposed mechanism was based primarily on mutagenesis experiments, isotope labelling and inhibitor studies. In a mutagenesis study by Sj/Sberg and coworkers, the Glu441 residue at the active site was substituted for Ala, Asp or Gln, whereby the explicit dependence of the Glu441 residue for catalytic turnover was revealed [55]. In addition, they could record the EPR spectrum of a new transient radical intermediate, most likely localized to the 3' position of ribose. From this work it hence became clear that not only the three cysteine residues were required, but also Glu441 providing H-bonding interaction and serving as "proton shuttle" (see below). In addition, the radical-based catalytic mechanism could definitely be established. Based on a large set of model calculations, Siegbahn revised and extend the Stubbe mechanism to invoke both the Glu441 residue as well as the conserved Asn437, hydrogen bonded to Glu441, in the catalytic machinery. The initial reaction in this 6-step mechanism is the abstraction of the C3'-H of ribose by the Cys439 radical formed through the radical transfer mechanism (Figure 25). The subsequent step involves simultaneous loss of O3'-H to Glu441, formation of a O3'-C3' double bond, transfer of the radical site to C2' of ribose, loss of the C2'-OH group and formation of water from the C2' OH group and the carboxylic hydrogen already bound to Glu441. Stabilizing H-bonding interactions to Asn437 is crucial for this complex sequence to proceed with low barrier. Next, the two cysteine residues C225 and C462 come into play. C225 looses its hydrogen to the C2' radical site of the substrate, C462 donates its hydrogen to the C225 thiol radical which forms a complex to the C3' position, and the 0 2 ' hydrogen is returned from Glu441. According to the computed energetics, this step is rate limiting. In step 5 C225 is released from the sugar to form a disulfide bridge between C225 and C462, and the radical character is transferred back to the C3' position. The final step involves back-transfer of the H-atom to C3', initially taken by Cys439. We have now formed the deoxyribose substrate, water and a disulfide bridge. The radical character at C439 is transferred back to Tyr122 of the R2 subunit while the substrate leaves, and the disulfide bridge is reduced through a sequence of coupled enzymatic reactions to regenerate the active site for another turnover. The mechanism is displayed in Figure 25, and in Figure 26 we show the overall energetics of the mechanism. It should be said, though, that the model has obtained some criticism, albeit it does

176

seem to fulfil essentially all experimental observations to date - including the abovementioned mutagenesis data for Glu441. PPO........~

?

~

H

S ,.,

PP*'~

Base

ase

H

H"-.-,,)~~H ./ \

SH '

o~

H

,

'

SH Step 1

I

I

H

C~

"O

SH

~

.~.~~H

o,

o..

H

,

C4~

',

SH

SH

C226

C41~

I

H.

i

I

Ste~

"O

Lo,?:

LH--- N

PPO-...~

Base

C

Step 2

PPO-......,/o

C. Ht,

H

siH

x.

SH

9

// 0

~"H

H /

,,0., H H

O

~o:

9

9

SH

I

I

C~

C4e=

Base

Step3

oH

"/--H

//

(--H

o

.

I

I

O.,

C=2s

C,~

O

..H

,

H

SH

"O H-- N I H

o

PPO"~

Base

co

+ + --

Step 4 _ S" S"

+SH

~

Base

,.~t__~ H

Step 6=

w

I ---..- I

,9

Czm C4=

H--O

.S

O"

H"

O." H - "O-,H.

~.

9S

"H-.--a,, 6 N

c.I

O- H - -

S

o'"

"

I

' 'S

I

h

6

c=,

c,=

:N---U,

N4wt

N

PPO~ ?'~ Se

Step 6

H

O

~C22s

1~ E44

ster~ 4

9

"o

/ H

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H"

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Base H

H

H

H--O O

1~ E~

o-H-" S O

-"

h

H

',

I

;

c=,

S

I

c,=

N,~

Figure 25. RNR class I reaction scheme as proposed by Siegbahn (comp.) [6].

177

Relative

kcal/mol 10

Energy

step 1 -

/+712/+2. 2 step 2 0 . 0 0 J +2.5\

HO

-10

OH

step3

step 4,,

~

-

-20

-

O

/

~-10.9 /

/

o

H

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" H

/ DNA

Figure 26. Overall energetics for catalytic mechanismof Class I RNR's [6]. The key intermediates of the sugar moiety are shown - labelling of different steps refers to Figure 25.

6. Concluding Remarks We have in the present chapter shown results from theoretical model system studies of the catalytic reaction mechanisms of three radical enzymes Galatose oxidase, Pyruvate formate-lyase and Ribonucleotide reductase. It is concluded that small models of the key parts of the active sites in combination with the DFT hybrid functional B3LYP and large basis sets provides a good description of the catalytic machineries, with low barriers for the rate determining steps and moderate overall exothermicity. The models employed are fin'thermore able to reproduce all the observed features in terms of spin distributions and reactive intermediates. For the Cu-containing system Galactose oxidase, we conclude that the unpaired spins initially are located on the Cu atom and on the axial tyrosine, whereas the equatorial, cysteine-linked tyrosine obtains the unpaired spin upon proton transfer from substrate to axial Tyr. The computed barrier for the rate determining step (H-atom transfer from substrate to Tyr-S-moiety) is in excellent agreement with experimental data, whereas the charge transfer between Cu(II) and substrate ketyl anion is less exothermic than estimated experimentally. A question is however raised regarding the experimental estimates, based on the computed data and the overall net exothermicity of the substrate reaction. In Pyruvate formate-lyase, the active site contains a stable glycyl radical and two catalytically active cysteines. Two different proposed reaction mechanisms were tested, and it was concluded that the mechanism as suggested

178 by Kozarich et al is by far the energetically most favourable one, albeit a minor modification in terms of radical mediated transesterification is proposed. Again, the reaction is characterized by a rather low barrier for the rate determining step. In addition, the mechanism for oxidative degradation of this anaerobic enzyme is outlined, based on a large set of model calculations. In Ribonucleotide reductase, f'mally, the radical transfer mechanism between the stable tyrosyl radical in the R2 subunit and the cysteine residue at the R1 active site is outlined, and shown to primarily invoke a neutral H-atom transfer pathway, with very low barriers and thermoneutrality. In addition, the substrate mechanism is outlined, based again on a model slightly modified compared with the original experimental proposals. In addition, several other radical enzymes have been investigated theoretically by us and others, such as DNA photolyase, Cu amine oxidase and prostaglandine H synthase, but we have found it beyond the scope of the present chapter to include all of these.

Acknowledgements The following people are gratefully acknowledged for their active roles in the studies of the above systems" M. Pavlov/Wirstam, Dr. J. Gauld, and Profs G.T. Babcock, F. Maseras, P.E.M. Siegbahn and A. Gr~islund. The Swedish Natural Sciences Research Council (NFR) is gratefully acknowledged for financial support. We also acknowledge the supercomputing centers at the Royal Institute of Technology in Stockholm (PDC) and at Link6ping University (NSC) for generous grants of computer time and Profs. J. Knappe and W. Kabsch for providing us with the coordinates of PFL prior to release on PDB.

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