Reaction Intermediates in Oxygen Activation Reactions by Enzymes

Chapter 24

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Reaction Intermediates in Oxygen Activation Reactions by Enzymes Containing Carboxylate-Bridged Binuclear Iron Clusters 1

2

3

Boi Hanh Huynh , J. Martin Bollinger, Jr. , and Dale E . Edmondson 1

3

Department of Physics and Departments of Biochemistry and Chemistry, Emory University, Atlanta, GA 30322 Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802

2

The rapidfreeze-quenchmethod was used to trap intermediates in the reaction of O with the dinuclear iron(II) clusters in the soluble methane monooxygenase from Methylococcus capsulatus (Bath) and in the R2 subunit of the ribonucleotide reductasefromEscherichia coli. Mössbauer and EPR spectroscopies were used to characterize the trapped intermediates. For methane monooxygenase, the initial intermediate formed is a peroxodiiron(III) complex which undergoes further structural and electron reorganization to form a high-valent, formally diiron(IV), complex termed Q. In the case of R2, reaction of O with the diiron(II) center in the presence of reducing agents generates a mixed valent, formally Fe(III), Fe(IV), complex termed X, which is capable of oxidizing the proximal Y122 to its radical form with formation of the resting diiron(III) cluster. In this chapter, spectroscopic characteristics of these intermediates are presented. Structural and mechanistic implications are also discussed. 2

2

In the last decade, it has been recognized that a group of proteins containing carboxylate-bridged binuclear iron clusters form a class of enzymes which activate molecular oxygen for diverse biological functions. These include the hydroxylase component of the soluble methane monooxygenase (MMOH) (/, 2), R2 subunit of the Fe-containingribonucleotidereductase (5-5), stearoyl-acyl carrier protein Δ desaturase (6), toluene monooxygenases (7, 8\ xylene monooxygenase (9), phenol hydroxylase and alkane hydroxylase (10). This realization has stimulated increased interest in the study of the structures and oxygen reactivities of these diironcontaining enzymes. Particularly, extensive spectroscopic and kinetic investigations have been performed on the soluble MMO enzyme system and on the R2 subunit of E. coliribonucleotidereductase (7, 2, 11, 12). Applying rapid kinetic techniques, such as stopped-flow and rapid freeze-rapid quench methods, several key 9

© 1998 American Chemical Society In Spectroscopic Methods in Bioinorganic Chemistry; Solomon, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

403

404 intermediates formed dining the oxygen activation reactions by MMOH and R2 were identified and spectroscopically characterized (13-26). In this Chapter, we present the kinetics and spectroscopic properties of these reaction intermediates, point out their physical characteristics and discuss the structural and mechanistic implications. Emphasis is given on the similarities as well as the differences between these two proteins in their active site structures and oxygen activation mechanisms. Our goal is to identify the factors that control their mechanisms and specificities.


The Enzyme Systems and Kinetics of Their Reaction with Oxygen Ribonucleotide Reductase. Ribonucleotide reductase (RNR) is a group of enzymes that catalyze the reduction ofribonucloetidesto deoxyribonucleotides, the first committed and rate limiting step in the biosynthesis of DNA. Three classes of RNR have been chracterized: the iron-tyrosyl radical containing RNR (class I), the adenosyl cobalamin-dependent RNR (class II) and the anaerobic RNR (class III) (27, 28). The E. coli RNR belongs to the class I enzymes and is composed of two homodimeric subunits, Rl and R2. The larger subunit R l , composed of two 85.7 kDa polypeptides, contains the binding sites for substrate and allosteric effectors, and the redox active cysteine residues involved inribosereduction. The smaller subunit, R2, composed of two 43.4 kDa polypeptides, contains the catalytically essential tyrosyl radical (Υ122·) and the binuclear iron cluster. Crystallographic studies (29) of met R2 (with the Y122 radical reduced) reveal that the two Fe atoms of the diferric center are each six-coordinate. They are bridged by an oxo and a carboxylate group (El 15). In addition, each Fe is ligated by one nitrogen atom from a histidine residue, a water molecule and two oxygen atoms from protein carboxylate groups (Figure 1A). This binuclear center is buried within a four helix bundle and is 10 Â from the nearest protein surface. The EXXH amino acid sequence is repeated in the diiron binding site and this repeat of EXXH sequence is now recognized as the binuclear cofactor binding motif for this class of oxygen-activating enzymes (30). The residue Y122 is located approximately 5.3 Âfromthe nearest Fe site (Fe^) and the Fe-Fe distance is 3.3 Â. The two ferric ions in R2 are antiferromagnetically coupled to form a diamagnetic ground state (57). They are spectroscopically distinguishable and exhibit two well resolved Mossbauer quadrupole doublets (AEQ = 2.41 mm/s and δ= 0.45 mm/s for doublet 1 and AEQ = 1.62 mm/s and S = 0.55 mm/s for doublet 2) (25, 57). The unusually large AEQ of 2.41 mm/s observed for one of the ferric site may reflect an asymmetric ligand environment caused by the bidentate binding of D84. More recently, the x-ray crystallographic structure of the reduced R2 has been solved at 1.7-À resolution (52). Although the same two histidine and four carboxylate residues remain as ligands to the reduced binuclear cluster, substantial ligand rearrangement occurs in comparison to the diferric form; residue E238 undergoes a "carboxylate shift (55)" shiftingfroma terminal ligand of Fe to a bridging ligand between the two ferrous ions and the coordination mode of D84 changesfrombidentate to monodentate (See Figure IB). Most interestingly, both ferrous ions are four-coordinate, resultingfromthe loss of the μ-οχο group and B

In Spectroscopic Methods in Bioinorganic Chemistry; Solomon, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

405 a water molecule for each Fe, and the Fe-Fe distance increases to 3.9 Â. These structural alterations upon reduction of the binuclear iron center should facilitate the binding of 0 . A MCD study of the reduced R2 has suggested that one of the Fe is five-coordinate (34). Currently, this discrepancy between the two methods is not yet understood. It has been known for more than two decades that this complex cofactor of R2 can be reconstituted spontaneously in vitro by addition of ferrous ions to apo R2 in the presence of molecular oxygen (35). The reaction can be described by equation 1. Downloaded by NORTH CAROLINA STATE UNIV on September 28, 2012 | http://pubs.acs.org Publication Date: June 9, 1998 | doi: 10.1021/bk-1998-0692.ch024

2

2+

+

ApoR2[Y122-OH] + 2Fe + 0 + H + e" 2

3+

2

3+

> R2[Y122-0#, Fe -0 "-Fe ] + H 0

(1)

2

An "exogenous" reducing equivalent is required for this reaction since the reduction of 0 to water requires four electrons and there are only three available electrons 2

from the diferrous cluster and Y122. The physiological source of the fourth electron has not yet been identified. In vitro, however, the fourth reducing equivalent can be provided by chemical reductants, such as ascorbate or excess ferrous ions. By using the methods of stopped-flow absorption spectroscopy, rapid freezequench EPR and Môssbauer spectroscopies, the R2 reconstitution reaction (at 5 °C) has been investigated in detail (22-26). Kinetic datafromthe three methods indicate that the R2 cofactor assembly proceeds according to Scheme 1, when the reaction is carried out with excess F e or in the presence of ascorbate. A single reaction intermediate, called X, forms with a rate constant of ~8 s and decays with a rate constant of ~1 s" (23). The decay of X is concomitant with the formation of Υ122· as well as the production of diiron(III) cluster. In the presence of excess Fe , the observed first order rate constant for formation of X is independent of the concentration of Apo R2, F e and 0 . This observation lead to the suggestion that conformational changes of R2 are required prior to its reaction with 0 to form X (23). In order to examine the validity of such a suggestion and also to investigate the reaction mechanism of reduced R2 with 0 , similar experiments were carried out 2+

_1

1

2+

2+

2

2

2

2+

2+

with Apo R2 preincubated with F e (precomplexed Fe -R2) before the introduction of 0 (20). With precomplexed Fe -R2, the same reaction intermediate X forms and decays with concomitant formation of Y122·, demonstrating that the cofactor assembly proceeds by a mechanism similar to that in apo R2. The formation rate of X, however, was found to increase by approximately 10 fold (60-80 s ) while the decay rate of X remains similar, supporting the suggestion of conformational changes prior to oxygen reaction (Scheme 1). 2+

2

-1

Methane Monooxygenase. Methane monooxygenase (MMO) catalyzes the conversion of methane to methanol (equation 2), the first step in carbon assimilation by the methanotrophic bacteria (36).



406

Figure 1. Schematic representation of the oxidized (A) and reduced (B) binuclear iron center of E. coli ribonucleotide reductase (adapted from ref. 30 and 33).


407 +

+

C H + 0 + H + NADH > CH OH +H 0 + N A D (2) Two classes of MMO have been identified, a membrane-bound Cu-containing enzyme and a soluble Fe-containing protein complex (57). The soluble protein complexes isolated from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b are the two most extensively studied MMO systems. They are composed of three protein components, a binuclear Fe cluster-containing hydroxylase (MMOH), an Fe S - andflavin-containingreductase (MMOR) and a small (16 kDa) metal free protein (MMOB). MMOH binds the substrate, and its binuclear Fe center is the site for hydroxylation. Kinetic investigations (14, 38-41) indicate that the reactivity of MMOH is regulated by both components MMOR and MMOB, most probably through complexation of the protein components involved (5c?). In particular, MMOB has been shown to have a significant effect on the 0 reactivity of reduced MMOH (40) as well as on catalysis by MMO (38, 41). Spectroscopic evidence further suggests that interaction of MMOB with MMOH affects the environment of the binuclear Fe center (38, 41-45). For a detailed account on the component interactions, the reader is referred to a recent review on this subject (1). The focus of this chapter is the transient intermediates formed during single turnover reactions of MMOH with 0 in the absence of substrate. The kinetics described below were results obtained from experiments performed with stoichiometric MMOB per binuclear site, an amount that has been shown to maximize the reactivity of MMOH with 0 . 4

2

3


2

2

2

2

2

2

MMOH is a large molecule of approximately 250 kDa comprised of three polypeptide chains organized in a (αβγ) configuration. The binuclear Fe cluster resides in the α subunit. As purified, MMOH is in the oxidized diferric form (H ). X-ray crystallographic measurements (46-48) show a similar structures for this center in M. capsulatus H flash frozen at -160 °C (46) and in M trichosporium H determined at 18 °C (48). Similar to the binuclear center of R2, the ferric ions are six-coordinate (Figure 2A), situated at the center of a four helix bundle and coordinated by two EXXH segments. Each Fe ion is ligated by a histidine residue. One of the Fe has two terminal monodentate glutamate ligands. The other Fe has one terminal glutamate and a water ligand. Unlike that of R2, in addition to a bridging carboxylate, the two ferric ions are bridged by two oxygen ligands forming a so called "diamond core" structure (75). In M. trichosporium H , the two oxygen atoms arefromtwo hydroxo groups and in M. capsulatus H , one oxygen isfroma hydroxide and the otherfroma water molecule. The Fe-Fe distance is short (-3.0 Â), which appears to be a characteristic for the diamond core structure (49, 50). Xray crystallographic studies (46, 47) further reveal that the binuclear center in M capsulatus H can exist in two structures depending on the conditions used in the Xray study. At 4 °C, the bridging water molecule is replaced by an exogenous acetate ligand (47), most probably derivedfromthe solvent. The Fe-Fe distance expands to 3.4 Â, a result probably reflecting the loss of the bridging water molecule. Recently, in an EXAFS investigation (57), two populations of molecules with different Fe-Fe distances were also found in a M trichosporium H sample. The majority (-60 %) 2

ox

o x

o x

o x

o x

o x


o x

408 of the sample exhibits a short Fe-Fe distance of 3.0 Â while the remainder shows a longer Fe-Fe distance of 3.4 Â. The presence of two structures for the binuclear ferric cluster has also been detected by Môssbauer spectroscopy in a single turnover M. capsulatus H sample (14). Two oxidized species, termed H (l) and H (2), are observed. H (l) is characterized by two equal intensity quadrupole doublets with AEQ =1.12 mm/s and 0.79 mm/s and £ = 0 . 5 1 mm/s and 0.50 mm/s, respectively, and H (2) exhibits two doublets with AEQ = 1.46 mm/s and 1.33 mm/s and δ= 0.72 mm/s and 0.47 mm/s, respectively. Kinetic Môssbauer data (14) indicate that these two oxidized species result from two populations of reduced MMOH, termed H (l) and H j(2), that react differently with oxygen; H (l) arises following a fast reaction of H (l) with oxygen while H (2) results from a much slower reaction of H (2) with oxygen. For the "as-purified" MMOH from both M. trichosporium and M. capsulatus, however, H (l) is the predominant species (45, 52). Currently, it is not clear whether the heterogeneity observed in the single turnover Môssbauer measurements can be correlated with that detected in the X-ray crystallographic and EXAFS studies. Upon reduction, the binuclear center of MMOH undergoes a substantial ligand rearrangement without changing the protein ligands (Figure 2) as found to be the case with R2. The terminal monodentate E243 in H (equivalent to E238 in R2) becomes a bridging ligand in H . Both exogenous bridging ligands are lost and the Fe atoms are five-coordinate. However, the bridging mode of E243 in H is distinct from that of E238 in reduced R2. Only one oxygen of the carboxylate of E243 is bridged between the two reduced Fe atoms. The other oxygen is still ligated to Feg (Figure 2). The Fe-Fe distance is 3.4 Â, which is significantly shorter than the 3.9 Â determined for reduced R2. This difference in the Fe-Fe distance could be partly due to the different bridging modes of E243 in H and E238 in reduced R2 and may be a factor that could influence the oxygen reactivities of the two enzymes. Another major structural difference revealed by X-ray crystallography that may be significant in substrate reactivity is the presence of a hydrophobic cavity adjoining the binuclear center in MMOH, which is not present in R2. Finally the carboxylate ligand E l 14 in MMOH differs from D84 in R2 by one carbon bond and may provide a better flexibility for the binding of Fe . o x

ox

ox

ox


ox

red

re(

ox

red

ox

red

ox

o x

r e d

r e d

r e d

A

The reaction of reduced M. capsulatus MMOH with 0 in the presence of 2 equivalents of MMOB and absence of substrate has been examined recently by a method combining rapid freeze quench technique and Môssbauer spectroscopy (14, 15). As mentioned above, two populations of H were found that react with 0 at different rates. H (l) (30-40 % of the total population) reacts with 0 at a rate (2428 s at 4 °C) that is about 100-fold faster than that of the enzyme catalytic turnover while H (2) reacts at a much slower rate (0.01 s" ) that is catalytically insignificant, suggesting that H (2) may represent non-functional enzyme. The 2

r e d

2

red

2

_1

1

red

red


409

ratio of these two populations was found to be preparation dependent. The origin for this heterogeneity is currently unknown. The kinetic Mossbauer and stopped-flow absorption data further establish that the reaction of H j(l) with oxygen proceeds according to scheme 2. Two reaction intermediates, H and compound Q, were found to accumulate at sufficient concentrations for spectroscopic characterization. Their spectroscopic properties will be described in the following sections. The first intermediate, H , accumulates at a rate (28 s ) that is consistent with the rate of disappearance (24 s ) of H j(l) and decays with the concomitant formation of compound β at a rate of 0.4-0.5 s . In the absence of substrate, compound Q decays with a rate of 0.03-0.07 s . The formation and decay rates of Q, monitored by stopped-flow spectroscopy, were found to be independent of oxygen concentration (14) suggesting the irreversible formation of a precursor in the reaction ofH (l)with0 . The oxygen reactivity of reduced MMOH from M trichosporium has also been investigated extensively by stopped-flow absorption and rapidfreeze-quenchEPR and Mossbauer spectroscopies (16, 17, 40). The results are consistent with those reported for H i(l) of the M. capsulatus enzyme. The spectroscopic properties, the formation and decay rates of the various spectroscopically detectable species are strikingly similar for these two enzymes. However, it is not clear whether the M trichosporium MMOH also contains two populations of molecules that react differently with oxygen. In the studies of the M. trichosporium enzyme, the decay of the reduced MMOH in its reaction with oxygen was monitored by the disappearance of the g = 16 EPR signal, which is characteristics of the diferrous cluster in reduced MMOH (53). Based on the observation that the rate of disappearance of the g = 16 signal is independent of oxygen concentration, it was proposed that an oxygen intermediate, called compound O, forms irreversibly prior to the formation of Hperoxo (40). Since the formation of Ο does not alter the g = 16 signal of the diferrous cluster, compound Ο was suggested to represent an oxygen adduct of MMOH where the oxygen molecule is not yet associated with the reduced binuclear cluster. re(

p e r o x o

-1

p e r o x o

_1


re(

-1

-1

red

2

rec

The Spectroscopic Detectable Transient Intermediates Hperoxo °f MMOH. The presence of an EPR silent, initial reaction intermediate in the reaction of MMOH with oxygen was first proposed for the M trichosporium enzyme based on the kinetic data obtained from stopped-flow optical and rapid freeze-quench EPR measurements (17). This transient intermediate, H , however was first spectroscopically characterized in the single turnover reaction of M. capsulatus MMOH with oxygen by Mossbauer spectroscopy (14, 15). It exhibits a sharp and symmetric quadrupole doublet with parameters AEQ =1.51 mm/s, S = 0.66 mm/s and a full-width at half maximum of 0.27 mm/s (Figure 3A). This same intermediate with identical Mossbauer characteristics has now also been identified in the M. trichosporium MMOH (13). At the time of its discovery, the observed p e r o x o


410

Η,Ο

0

Χ

\ J?L

/

0

Ε

209


"3ΙΑ~

Figure 2. Schematic representation of the oxidized (A) and reduced (B) binuclear iron center of the hydroxylase component of soluble methane monooxygenase (adaptedfromref. 47-49). k = fast H

red"

[Compound O]

k~25s-

k-O^s-

!

A

peroxo

1

Compound Q

k ~ 0.05 s-

Scheme 2.

-2

0

2

VELOCITY (mm/s)

Figure 3. Môssbauer spectra at 4.2 Κ of intermediates H (A) and compound Q (B) formed during the reaction of oxygen with reduced MMOH from M capsulatus. The reaction wasfreeze-quenchedat 155 ms (A) or at 8 s (Β). These spectra were preparedfromthe corresponding raw data by removing contributions of other iron species present in the samples from the total iron absorption (adaptedfromref. 16). p e r o x o


411

Mossbauer parameters were considered to be unique for carboxylate-bridged dinuclear Fe clusters. Based on the kinetic data and chemical considerations this intermediate was proposed to be a peroxodiferric complex even though the isomer shift of 0.66 mm/s was significantly larger than the 0.52-0.54 mm/s observed for the few peroxodiferric model complexes known at the time (54-56). The larger isomer shift of H can be explained as an indication of considerable electronic charge transfer from the peroxide to the Fe atoms. The fact that only one sharp and symmetric doublet was observed for H strongly suggested a symmetric binding mode for the peroxide. Mossbauer spectra of H recorded at strong applied fields (4 - 8 T) further demonstrated that the two ferric ions are antiferromagnetically coupled to form a diamagnetic ground state. A detailed stopped-flow optical absorption study of the reaction of M. trichosporium MMOH with oxygen has also shown that H exhibits a broad optical absorption band with « 725 nm (see the Chapter by S. J. Lippard in this book). p e r o x o


p e r o x o

p e r o x o

p e r o x o

Table I. Spectroscopic Properties of H

in M capsulatus M M O H and the

p e r o x o

Peroxodiferric Model Complex [Fe C"-0 )(//-02CCH2Ph)2{HB(pz')3}2] 2

2

a

H "peroxo

Diferricperoxo Complex

725

694

1.51

2650 1.40

^maxinm) -1

_1

ε (cm M ) AEQ (mm/s) δ (mm/s)

0.66

0.66 888 46

1

ν (O-O) (cm" ) 1

2

l (l6o -l8o )(cm- ) v

2

b

2

a

Data taken from references 14. Also, see the chapter by S. J. Lippard in this book. b

Data takenfromreference 57. pz' = 3,5-bis(isopropyl)-pyrazolyl.

Recently, a (//-peroxo)bis(//-carboxylato)diferric model complex (57) with a trans μ-η :η bridging peroxo structure has been reported to exhibit spectroscopic properties resembling those of H (Table I). In particular, the Mossbauer parameters of the model complex and H are strikingly similar, indicating that the complex is a potential structural model for the binuclear center in H . ι

ι

p e r o x o

p e r o x o

p e r o x o

Compound g. The transient intermediate, compound Q, was first discovered in the reaction of M. trichosporium MMOH with 0 (17). It is EPR silent and exhibits an optical spectrum with at 330 and 430 nm. The same intermediate with similar optical properties was also found later in the M. capsulatus enzyme (14) and was demonstrated to be the intermediate formed immediately after the decay of H . The 4.2-K zero-field Mossbauer spectra of compounds Q formed in M. capsulatus 2

p e r o x o


412 MMOH and M. trichosporium MMOH are very similar and show a quadrupole doublet with unusual parameters. However, for the M. trichosporium compound Q, the doublet is symmetric (16) and can be fitted with one set of parameters (AEQ = 0.53 mm/s and δ - 0.17 mm/s) while the M. capsulatus compound Q exhibits a slightly asymmetric doublet (Figure 3B) that is best fitted with two unresolved doublets (AEQ = 0.68 and 0.55 mm/s and S= 0.21 and 0.14 mm/s, respectively) (75). For a series of structurally related Fe compounds, the parameter, isomer shift, is a very good measure of the Fe oxidation state, with smaller isomer shift corresponding to higher oxidation state (

Reaction Intermediates in Oxygen Activation Reactions by Enzymes

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