Ortiz de Montellano Paul R.(Ed.) Cytochrome P450. Structure, Mechanism, and Biochemistry

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164

Thomas M. Makris et al.

Ala245Ser and Ala245Thr mutant enzymes, it

appears as though the loss of catalytic activity^^^

in the mutant forms correlates with the loss

or repositioning of Wat519 (ref [234]). None-

theless, these mutants are still able to metabo-

lize the substrate testosterone at different sites^^^,

implying that a very intricate hydrogen-

bonding interplay exists between substrate and

enzyme.

Molecular dynamics simulations performed by

Harris and Loew^^ provide additional evidence

regarding the role of active-site hydrogen bonding

in CYP107. By subjecting the peroxo-anion

species in the substrate-bound active site of the

enzyme to molecular dynamics simulation, two-

proton-donating partners, the C5 hydroxy 1 of

the bound substrate, and a bound water Wat519

are seen to form-up hydrogen bonds on the distal

oxygen atom of the ligand within 25 ps from

initiation of the simulation^^. Furthermore, con-

nectivity of this hydrogen-bonding network to

other exchangeable groups, namely to Wat564,

Ser246, and Glu360 suggests a specific proton

relay pathway in the enzyme.

In order to properly assess the molecular basis

of peroxide forming chemistries in the Thr252X

mutants, one must first adequately assess the

operant chemical mechanism or mechanisms that

give rise to the observed uncoupling. As described

earlier, the use of cryogenic irradiation has proven

to be an invaluable tool in dissecting the particular

intermediate state involved in both P450 mono-

oxygenation and peroxide evolving chemistries.

Using low temperature radiolytic reduction of the

Tlir252Ala mutant ferrous-oxy complex, the

enzyme directly forms the protonated peroxo

anion"^^'

as is readily identified by the character-

istic shift in g-tensor upon protonation of the

distal oxygen*''^' '^^. This result is congruent

in terms of the unmodified rates of pyridine

nucleotide oxidation seen in room temperature

studies. First, despite mutation, the Thr252Ala

mutant of CYPlOl, like the wild-type enzyme, is

able to protonate the distal oxygen atom of the

peroxo-anion species. This in turn, in the course of

temperature annealing, decays to the ferriheme

resting state with no intervening intermediates.

In order to ftirther assign the hydroperoxo-ferric

species as the branchpoint of peroxide forming

and monooxygenation studies, analogous experi-

ments were performed with the double mutant.

Thr252Ala/Asp251Asn, in which the stoichiomet-

ric ratio of the two pathways is equal at room

temperature

^^^.

As primary proton transfer is also

visible at low temperature in this mutant con-

struct, prior to decay to the resting state, one is

able to definitely assess that the protonated peroxo

intermediate serves as the branching intermediate

of these two pathways.

4.2.

The "Conserved" Acid

Functionality

As in the case of the conserved hydroxyl

moiety in the P450 active site, a conserved acid

residue has similarly been implicated as the criti-

cal proton transfer vehicle in the P450-catalyzed

oxygen activation. Upon mutagenesis of Asp251

in P450 CYPlOl to asparagine, a phenotype quite

distinct from that of the alcohol mutagenesis is

observed^^'

^^K

Instead of the normally high, but

decoupled, rate of pyridine nucleotide consump-

tion seen in the Thr252Ala mutant, the enzymatic

turnover activity of the Asp251Asn mutant

is decreased by more than one order of magni-

tude^^'

^^.

With close examination of the separate

catalytic parameters of the P450 reaction wheel

(Figure 5.2), this decreased activity was specifi-

cally localized to second electron transfer and

linked protonation events following formation of

the ferrous-oxy complex^^' •^^. Unlike the con-

served hydroxyl moiety in the immediate vicinity

of the heme iron, a number of mutations at this

site have revealed that the observed phenotypical

variance is poorly explained by a discrete physic-

ochemical property of the amino acid side chain

that is inserted. For example, both the

Ala251

and

Gly251 substitutions behave similarly to that of

the isosteric charge reversal Asp251Asn^^^. While

these phenotypical consequences of Asp251

mutation are similar throughout many P450

sequences, such as CYP107, the kinetic efficiency

of the mutant enzymes often depends on the iden-

tity of the substrate being metabolized'^^'

^36-238

Some indication of active-site acidic ftinction-

ality in P450 dioxygen scission can be revealed by

the comparative evaluation of kinetic parameters

in protonated and deuterated waters. The use

of kinetic solvent isotope effects (KSIEs) has been

valuable in the determination of potential proton-

linked dioxygen activating enzymes such as

P45038,

39^ methane monooxygenase (MMO)^^^,

Activation of Molecular Oxygen by Cytochrome P450

165

and the cytochrome oxidases^'*^. It is important

to prove that the observed isotopic effects are

specifically linked to proton transfer events in the

dioxygen cleavage reaction. In the case of cyto-

chrome P450 CYPlOl, detailed kinetic studies

demonstrated that only the second electron trans-

fer rate, and the linked concomitant catal3^ic steps

were sensitive to solvent isotope content^^. This in

turn has allowed the comparable gauging of iso-

topic sensitivity of the dioxygen cleavage reaction

simply through the rate ratio measurements of

product formation. Through the comparative

analysis of KSIEs and proton inventories of the

wild-type and the Asp251Asn mutant CYPlOl

enzymes, a startling difference in these parame-

ters is observed^^. In the wild-type enz3ane, by

fitting the proton inventory to a range of fraction-

ation factors, it was shown that at least two

protons are in flight during the observed transi-

tion state process. This is consistent with the

previously discussed schemes involving distal

pocket waters and the hydrogen-bonding Thr252.

Furthermore, a 4-fold increase of the KSIE in the

Asp251Asn mutant, as well as linear dependences

of NADH oxidation rate on bulk proton concen-

tration, intimates that the proton delivery conduit

in this mutant is drastically altered, perhaps medi-

ated by a chain of water molecules^^.

In structural terms, crystallographic analysis of

the Asp251Asn mutant has provided additional

evidence for its involvement in proton delivery^^.

In the wild-type ferric structure of CYPlOl,

the Asp251 side chain itself points away from the

distal pocket, and instead interacts in a bifur-

cated salt-bridge involving Argl86 and Lysl78.

Although other structural features of the distal

pocket remain intact, mutagenesis to Asn251

results in the simultaneous rotation of

the

Asn251

and Lysl78 side chains to the protein surface,

resulting in loss of their intermolecular contacts.

This in turn is compensated by a new interaction

of the

Asn251

side chain with Asp

182.

These mul-

tiple motions speak to a highly plastic active site

with a resulting open access of the heme iron from

the protein surface. While it is difficult to translate

the observed KSIE and pH dependence data of the

Asn251 single mutant to a rigorous structural

model, in part due to difficulty in visualizing

active-site mobile waters, it nonetheless remains

consistent with a proton delivery mechanism

modulated by additional solvent molecules.

As in the case of the Ala252 mutation, the

annealing profile of cryogenically reduced oxy-

ferrous

Asn251

mutant in P450 CYPlOl corrobo-

rates the kinetic data just discussed. Unlike the

wild-type and Ala252 mutant enzymes, which

form the hydroperoxo-ferric complex upon irradi-

ation at 77 K, EPR g-tensors and ^H ENDOR

couplings reveal that the primary reduced com-

plex of Asn251 is the unprotonated peroxo-anion

species"^^'"^^ Upon annealing to the glass transi-

tion temperature (190 K), protonation of the

peroxo anion occurs, confirming that the pheno-

typic effects seen in this mutant are localized

at primary proton delivery. Moreover, the

temperature-dependent annealing profile likewise

implies an active-site conformational change in

the mutant protein is required to enable efficient

proton transfer. This reorientation of the bifur-

cated salt-bridge during CYPlOl catalysis has

additionally been implicated by the dependence

of turnover rate on hydrostatic^'*^ and osmotic^'*^

pressures, as well as detailed photoacoustic

calorimetry measurements^"^^'

-^^^

of both wild-

type and mutant cytochromes. These results

implicate a distinct and localized re-solvation of

active-site residues during substrate turnover.

However, localization of these dependences to

protonation of

the

peroxo anion is complicated as

it is mixed with analogous reorientations and

conformational changes occurring during other

catalytic steps such as substrate binding. Never-

theless, the implication of a rotameric flexibility

of the Asp251 side chain, stabilized by discrimi-

nating electrostatic interactions, presents an

important theme in the development of a dynamic

model of P450 dioxygen scission.

4.3.

Crystallographic Studies

of P450 Reaction

Intermediates

As with spectroscopic characterization of the

peroxo-ferric intermediates in CYPlOl, one of

the most revealing studies of P450 dioxygen activa-

tion has been the recent structural resolution of

P450 intermediates using cryocrystallography^^.

This work not only provided a molecular picture of

P450 intermediate states, but also a road-map for

the participation of the distal pocket in the effective

delivery of protons to a ferrous-dioxygen species.

166

Thomas M. Makris et al.

Analogous to the spectroscopic work outlined in

previous sections, these studies rely on cryogenic

radiol)^ic reduction in the form of exposure to

long-wavelength X-rays in order

isolate normally

reactive heme-oxygen intermediates. However, in

contrast to the spectroscopic analysis of low-

temperature reduced samples, the X-ray crystallo-

graphic analysis of the X-ray reduced sample is

complicated by the fact that the X-rays not only

serve as a pulse but also as probe. Therefore,

sample radiolysis cannot be totally controlled or

eliminated. Diffusion of dioxygen into dithionite-

reduced crystals of CYPlOl at low temperature,

and using short wavelength X-rays to mini-

mize radiolysis, revealed the heretofore structurally

uncharacterized P450 ferrous-dioxygen complex.

These structural studies nicely complement the

low-temperature spectroscopic characterizations.

Structural resolution of oxy-ferrous P450

reveals important differences from the ferrous

precursor. The accompaniment of dioxygen

ligated in an end-on

(T^J

coordination geometry) to

the heme-iron correlates to a number of structural

nuances. First, and perhaps most obvious, is the

presence of two new water molecules, WAT901

and

WAT902,

in the active site of the enzyme. One

these,

WAT901, appears both within hydrogen-

bonding distance of the dioxygen ligand and the

Thr252 hydroxyl side chain (Figure 5.4). This

water molecule is unique to the ferrous-dioxygen

structure, but may in fact represent the stabilized

water implicated in proton delivery in the threonine

mutagenesis and solvent isotope-effect studies out-

lined above. In addition, the backbone amide of

Thr252 occupies a "flipped" orientation allowing

not only additional stabilization of this bound water

molecule through new hydrogen-bonding interac-

tions with the peptide backbone, but also a new

inter-action of the

Asp251

backbone carbonyl with

the amide and amino groups of

Asn255.

Figure 5.4. Overlaid crystal structures of the ferrous (light) and oxy-ferrous (grey) wild-type CYPIOI.

Activation of IVIolecular Oxygen by Cytochrome P450

167

The observation of this new hydrogen bond

network in the crystals of the ferrous-dioxygen

complex provides a satisfying structural explana-

tion of the observed changes in coupling and

turnover upon mutation of Asp251Asn and

Thr252Ala. In the case of the latter, the threonine

hydroxyl, in addition to the backbone amide,

donates key stabilization interactions with both

bound dioxygen and

WAT901.

One might expect

that the removal of these interactions in the

Thr252Ala mutant would have effects at multi-

ple branchpoints of the P450 catalytic cycle.

Elimination of this H-bonding interaction would

result in a relative destabilization of the oxy-

ferrous complex, resulting in an increased rate of

autoxidation in the Thr252Ala mutant^^^ In addi-

tion, the newly observed water molecule in the

ferric Thr252Ala structure, WAT720, is localized

about

A further from the dioxygen moiety than

in the wild-type oxy-ferrous counterpart. While

efficient primary-proton transfer is observed in

this mutant in radiolytic studies, the crucial proton

transfer step, resulting in water formation and

heterolytic cleavage to form "Compound I," is

perturbed by the modification of these inter-

actions. Interestingly, the flip of the Asp251

carbonyl is also seen in the Thr252Ala ferric

structure, although the rotation is enhanced even

more (120° vs 90°).

The structure of the CYPlOl wild-type

ferrous-dioxygen complex also provides insight

into the diminished efficacy of primary proton

delivery in the Asp251Asn mutant, as observed in

both room temperature kinetics and EPR annealing

profiles. The "flip" of

the Asp251

backbone is syn-

ergistically coupled with the rotation of the Thr252

backbone. In the ferric structure of Asp251Asn,

the mutated

Asn251

side chain forms new interac-

tions with Asp 182 and Asn255, with the latter

effectively blocking the interaction with the 251

carbonyl in the "flipped" conformation typical for

the oxy complex. Examination of the oxy-ferrous

structure of Asp251Asn corroborates this finding,

as neither of the catalytically critical solvent mole-

cules is observed (Schlichting et ai, unpublished).

The differential solvation of the active site in

the Asn251 and Ala252 mutants is also observed

in resonance Raman spectra of the ferric cyanide

adducts^"^^. Specifically, while the wild-type

enzyme shows exclusive sensitivity to isotopic

exchange in the bent conformer, those of the

mutants show spectral changes upon D2O

exchange in both linear and bent conformations.

As the bent conformer better represents the

ferrous-oxy complex as observed in structural

studies, one would expect that it is exclusively

subject to isotopic exchange in the wild-type

enzyme. Meanwhile, as mutation would cause

drastic changes in hydrogen-bond patterning in

the distal pocket, it is reasonable to expect iso-

topically induced vibrational changes of both con-

formers in the mutants. Crystallography of the

cyanide complex of wild-type CYPlOl, in which

the bent conformer is observed, illustrates the

interaction of the cyanide ligand with both the

threonine hydroxyl and

WAT901

(ref [246]).

By irradiating oxy-ferrous P450 crystals with

longer X-ray wavelengths, catalytic processing in

the active site can be initiated and structurally

resolved. Radiolytic reduction of the oxy-ferrous

crystal of CYPlOl results in distinct changes

localized in the distal pocket. These are per-

haps best illustrated in overall electron density

difference maps"^^. Most importantly, following

introduction of an electron to the preformed oxy-

complex is the appearance of a negative difference

density localized at the distal oxygen atom of

the iron-bound dioxygen. This suggests that oxy-

gen-oxygen bond cleavage has occurred leaving

behind a single oxygen atom ligated to the heme

iron. The resulting Fe-O bond distance appears

shortened to about 1.67 A, consistent with an iron-

0x0 "Fe=0" type structure in analogy with that

observed in CcP (1.66

A)^^^,

HRP

(1.70 Ay^\ and

catalase (1.76 A) (Protein Databank entry IMQF).

In these cases, the iron is seen to move to a

position slightly above the heme plane. Frustrat-

ingly, despite numerous attempts, the resulting

"ferryl-oxo" structure does not have a fully occu-

pied oxygen atom and could not be precisely

refined without constraints. Nonetheless, the

resulting picture is extremely exciting as it shows

that the twice-reduced iron-oxygen complex is

catalytically competent. Indeed, warming the

crystal to a point above the glass transition tem-

perature reveals a structure with clear positive

electron density corresponding to a 5-exo alcohol

hydroxycamphor product. In the product complex,

the protein displays the resting state conformation

with respect to water and protein structure. No

"extra" water molecules appear in the active site

and the Thr251-Asp252 backbone conformation

168 Thomas M. Makris ef a/.

has moved back to that found in the ferric and

ferrous camphor-bound complexes.

Further differences between studies performed

on frozen solutions and crystals can be attributa-

ble to crystal contacts that may have a crucial

impact on the dynamics of the system, thereby

changing relative rates of subsequent reaction

steps.

For example, the lack of accumulation of

[6] in cryoradiolytically reduced frozen solu-

tions'*^'

but apparently not in crystals'*^ may be

related to a crystal contact involving the potas-

sium binding site and the carbonyl oxygen atom of

Tyr96 in the monoclinic crystal form of CYPlOl

(Figure 5.5). The hydroxyl group of Tyr96 forms a

hydrogen bond with the keto group of camphor,

thereby controlling its position, mobility, and

thus the distance between the C5 atom of camphor

and the reactive oxygen species.

Given the dynamic nature of both intermediates

and the isomerization of amino acids of side chains

occurring during catalysis, it is not surprising that

a number of computational studies have provided

subtly different structural mechanisms for the

acid-alcohol pair in P450 catalysis. Molecular

d3niamics simulations of the ferrous-dioxygen

complex, prior to its structural resolution described

above, suggested a possible role for Thr252 (ref

[92]).

During the course of the simulation, the

Thr252 hydroxyl, instead of interacting with the

Gly248 backbone as modeled initially, prefer-

entially participates in a hydrogen-bonding inter-

action with the distal oxygen atom. While this

interaction persists throughout the simulation,

no significant movement of the Asp251 residue

was observed. A more direct role for the con-

served acid residue in protonation events has

very recently been made by Hummer and col-

leagues^"^^. Through the examination of allowable

side chain and water fluctuations, utilizing the

ferrous-dioxygen state as a precursor, an interest-

ing model for the active-site modulation of proton

networks emerges. The authors note that the

movement of the Asp251 side chain toward heme-

bound dioxygen is almost unrestricted and that the

correlated movement of this residue and WAT901

provides effective proton connectivity between

these two moieties. The addition of

one

new water

molecule links this local network to the surface of

the protein and bulk solvent. While such a

dramatic rotation of the Asp251 side chain was

molecule 2

molecule 1

Figure 5.5. A crystal contact at the potassium binding site may influence the dynamics of active-site moieties,

particularly Tyr96, and thus indirectly, camphor.

Activation of Molecular Oxygen by Cytochrome P450

169

not observed in static crystal structures of resting

or dioxygen bound form, such localized confor-

mational changes can obviously significantly

modulate proton delivery^^^

4.4. Mechanism-Based

Specificity of Proton

Transfer

Conformational changes in the distal pocket

reorientation can result in drastic effects on the

concerted delivery of protons to heme-bound

dioxygen. Coupled with these movements is the

mechanism-based specificity of proton transfer as

determined with the identity of the substrate to

the specific P450. The site directed transforma-

tion of various heme enzymes to accommodate

novel reactivities, both in substrate recognition

and through mechanistic inferences of proton

delivery, has led to a successful redesign of pro-

tein peroxo-iron chemistries. These have been

documented in histidine- and thiolate-ligated per-

oxidases^^^"^^^. As a feat in reengineering, the

directed alteration of reactivity is perhaps best evi-

denced in the redesign of the dioxygen carrier

myoglobin, normally defunct with regards to het-

eroatom oxygen transfer reactions, to evolve suc-

cessful monooxygenation chemistries utilizing

both hydrogen peroxide^^^'

252-254

^^^ dioxygen^^^

as the source of oxidant. Within the family of

P450 enzymes, two examples of altered reactivity,

that of nitric oxide reductase and peroxygenase

activities, illuminate mechanisms of active-site

complementarity to a specific ligand.

The role of proton transfer in P450-catalyzed

nitric oxide reduction has been implicated in a

number of computationaP^^ and experimental

studies^^^, even though a complete mechanistic

assignment of particular nitrogen ligated states

remains elusive. Structural analysis of the

P450nor active site from Fusarium oxysporum by

Shiro and colleagues reveal that, unlike CYPlOl,

the active site is comprised of a number of

hydrophilic residues^^^"^^^. Although the "con-

served" threonine is localized in the distal pocket

(Thr243), the orientation of its side-chain

hydroxyl is subtly different, with the alcohol side

chain pointing toward the dioxygen-binding site.

Whether this reflects a more natural rotamer con-

figuration not seen in the other P450s structurally

examined, or if significantly different mechanisms

are present is unclear. Similar differences in the

relative positioning or flexibility of the threonine

have likewise been inferred in the examination of

lysine mutants in various P450s^^^' ^^^. The con-

nectivity of the threonine hydroxyl in P450nor

with the I-helix main chain is retained, however,

through a solvent molecule, WAT46, to the pep-

tide carbonyl of the i-4 residue Ala239.

Meanwhile, the threonine is involved in another

water channel leading to the distal face, and

finally bulk solvent. Such a change in polarity

of the distal pocket between P450nor and CYPlOl

is transmitted even into the observed coordina-

tion geometry of bound NO and isocyanide

complexes^^^'

^^^.

Mutagenesis of the Thr243 residue in P450nor,

like the Thr252, results in a drastic reduction

of activity, in this case the NADH dependent

reduction of NO to N2O (ref [265]). Nonetheless,

structural analysis of several mutants at this

position show very minor perturbations of a plau-

sible proton delivery network^^^. Thus, while the

appearance of new water molecules in mutant

forms is certainly correlated with a change in a

possible proton delivery network, it is difficult to

eliminate the possibility that the residue could be

functioning by facilitating electron transfer from

NADH, as the enzyme directly catalyzes electron

transfer from pyridine dinucleotide to the active

site.

This potential alteration in the role of a con-

served threonine in proton delivery is likewise

supported by the absence of a conserved acid

residue, Ala at position 242, in P450nor, and may

reflect an alteration of the distal pocket network to

accommodate its unique mechanism. Based on

its closer proximity to the NO binding site and

similar abolishment of activity upon mutation,

another hydroxyl, Ser286, may instead fulfill

the role of the conserved P450 hydroxyl^^^' ^^'^.

Unlike the Thr243 mutants, structural analysis of

various Ser286 mutants shows drastic changes

in hydrogen-bonding networks through the sys-

tematic destabilization of water networks^^^' ^^^.

The effects of these water networks involv-

ing Ser286 appear critical, as even the minor

Ser286Thr mutation results in a rotamer con-

figuration, which destabilizes the water and

results in a loss of activity. Thus, the active-site

proton delivery networks of these heme-oxygen

enzymes could be very plastic with the precise

dynamical picture of hydrogen ion delivery not yet

revealed.

170

Thomas M. Makris et ai.

While P450s are typically poor in the utiliza-

tion of hydrogen peroxide as a dioxygen/elec-

tron/proton surrogate in monooxygenation

chemistry, a number of P450s have been shown to

utilize the peroxygenase pathway in the hydroxy-

lation of fatty acids^^^"^^^ Evaluation of these

enzymes is important in order to assess the

isozyme dependence of various P450s in the uti-

lization of the peroxide shunt pathway in catalytic

processes. In order to accommodate this funda-

mental difference in active-site chemistry with

these P450s, as in this case both protons and elec-

trons are delivered to the ferriheme moiety, the

enzyme is devoid of the conserved acid-alcohol

pair. For example, in P450 CYP152A1 these posi-

tions in the active site are occupied by Arg242 and

Pro243 respectively. However, mutagenesis has

succinctly demonstrated their critical role in both

fatty acid binding and in the hydrogen peroxide

mediated hydroxylation reaction^^^. Likewise,

both the presence and positioning of a substrate

carboxylate is similarly crucial presumably in

an electrostatic interaction with this conserved

arginine in peroxygenase^^^'

^'^^.

Recent crystallo-

graphic structure determination of this P450

by Shiro and coworkers have suggested a very

elegant mechanism of substrate mediated peroxy-

genase catalysis^^^. The fatty acid carboxylate is

found in a similar orientation as the Glul83

residue in chloroperoxidase^^^ and thereby seems

to serve as the catalyst for O-O bond scission

of hydrogen peroxide^^'. While the conserved

histidine found in chloroperoxidase is absent in

the peroxygenase distal pocket, the role of modu-

lating the basicity of the substrate carboxylate

may be served by Arg242 (ref [275]).

4.5. Summary

The rich mechanistic enzymology of the

cytochrome P450s has occupied chemists, bio-

chemists, pharmacologists, and toxicologists for

over three decades. Are we near to a detailed

molecular understanding? We have attempted to

convey in this chapter of Cytochrome P450 the

recent discoveries that fill many of the lacunas in

our understanding of P450-catalyzed substrate

oxidations. We now have a precise three-

dimensional structure of the ferrous-oxygenated

state,

and ample spectroscopic characterization of

the ferric-peroxo anion and ferric-hydroperoxo

intermediates. In the exogenous oxidant driven

pathway, an archaeal P450 allowed facile obser-

vation of the formation and breakdown of

the "Compound I" ferryl-oxo state. Yet much

remains. Stabilization and characterization of the

"Compound I" state in the dioxygen reaction has

not yet been achieved. With the ability to separate,

through time and temperature, the population of

multiple "active oxygen" intermediates in P450

catalysis, it remains to precisely define the reac-

tivity profiles of each state and thereby realize

a mapping of observed in vivo metabolic profiles

to specific states in the reaction cycle. An over-

riding revelation has been the subtle way in which

Nature controls the reactivity of atmospheric

dioxygen, electrons, and transition metal through

delicate hydrogen-bonding interactions. Thus,

in a Periclesian control of mechanism, the cyto-

chromes P450 utilize a variety of proton pathways

to finely tune this versatile catalyst for critical

physiological processes.

Acknowledgments

This work was supported by National Institutes

of Health grants R37 GM31756 and GM33775

(SGS) and the German Research Foundation (IS)

grant BIO2-CT942060 (IS).

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