PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Am Chem Soc. Author manuscript; available in PMC 2010 May 6.
Published in final edited form as:
PMCID: PMC2745944
NIHMSID: NIHMS109233

Characterization of a Peroxodiiron(III) Intermediate in the T201S Variant of Toluene/o-Xylene Monooxygenase Hydroxylase from Pseudomonas sp. OX1

Abstract

We report the observation of a novel intermediate in the reaction of a reduced toluene/o-xylene monooxygenase hydroxylase (ToMOHred) T201S variant, in the presence of a regulatory protein (ToMOD), with dioxygen. This species is the first oxygenated intermediate with an optical band in any toluene monooxygenase. The UV-Vis and Mössbauer spectroscopic properties of the intermediate allowing us to assign it as a peroxodiiron(III) species, T201Speroxo, similar to Hperoxo in methane monooxygenase. Although T201S generates T201Speroxo in addition to optically transparent ToMOHperoxo, previously observed in wild type ToMOH, this conservative variant is catalytically active in steady state catalysis and single turnover experiments, and displays the same regiospecificity for toluene and slightly different regiospecificity for o-xylene oxidation.

Carboxylate-bridged diiron centers are common active site motifs in enzymes, performing a variety of dioxygen-dependent functions, including hydrocarbon oxidation,14 The diiron centers in these enzymes have similar primary coordination spheres,57 suggesting that the protein scaffold tunes their chemical reactivity to favor a specific reaction pathway. Previous construction of variants of RNR-R28 and ToMOH demonstrated that changes in the secondary and tertiary coordination spheres can divert the reaction pathway from electron abstraction to aromatic hydroxylation and vice versa.9,10 For these variants, however, the chemistry of the diiron(II) cluster with dioxygen was unchanged, and the oxygenated intermediates reacted with nearby amino acid residues. Here, we provide the first direct evidence of that a single conservative amino-acid mutation in BMMs partially bifurcates the oxygenation reaction to generate a diiron(III) peroxo intermediate not observed in the native system.

Recently, a transient diiron(III) intermediate in ToMOH, ToMOHperoxo, was identified by Mössbauer spectroscopy and shown to be kinetically competent for arene oxidation.11 Although this enzyme is similar to sMMOH both in sequence12 and active site structure,13 the spectroscopic properties of ToMOHperoxo were unexpected. Whereas Hperoxo in sMMOH displays an optical band at ~ 720 nm (ε720 = ~ 2000 cm−1 M−1)14,15 and exhibits Mössbauer parameters of δ = 0.66 mm/s, ΔEQ = 1.51 mm/s,16 ToMOHperoxo has no optical bands in the visible region and Mössbauer parameters of δ = 0.54 mm/s and ΔEQ = 0.67 mm/s.11 No other transient intermediate, like Q, was observed in ToMOH. These results suggest that ToMOHperoxo has a structure distinct from that of any other peroxodiiron(III) intermediate in carboxylate-bridged diiron proteins, including sMMOH, Δ9D,17 and RNR-R2 variants.18,19

Here we report the observation of a novel peroxodiiron(III) intermediate in a variant of ToMOH (T201Speroxo) having spectroscopic properties similar to those of Hperoxo in sMMOH. The T201S variant (hereafter T201S) was originally prepared to investigate the role of a strictly conserved threonine residue near the carboxylate-bridged diiron active site on catalysis.13 During the course of our study, a structure of the T4moHD complex was reported revealing T201 to be involved in a novel hydrogen-bonding network terminating at a water molecule coordinated to Fe1.20 The steady state activity for conversion of phenol to catechol in T201S was measured to be 2400 ± 300 mU/mg (mU = nmol/min), compared to that of wild type enzyme, 1200 ± 200 mU/mg, respectively.21 This result indicates that T201S is even more efficient than wild type enzyme in aromatic hydroxylation. A Michaelis-Menten kinetic analysis revealed kcat and kcat/KM values for T201S of 0.08 ± 0.03 s−1 and 0.02 1µM−1s, respectively, which are not greatly different from those of the wild type enzyme, 0.049 ± 0.003 s−1, and 0.011 ± 0.003 µM−1s−1. The T201S variant produced the same product yield as wild type enzyme, corresponding to ~ 50% of the diiron sites in phenol oxidation in a single turnover reaction of reduced diiron(II) TOMOH with O2.22 The regiospecificity of T201S for toluene hydroxylation was also conserved at a 3:2:5 ratio of o:m:p-cresol, and that for o-xylene hydroxylation was slightly perturbed in T201S, changing the ratio of the two products from 2:8 to 4:6 2,3-dimethylphenol vs 3,4-dimethylphenol.23

When investigating pre-steady-state dioxygen activation at the reduced diiron(II) center of T201S by stopped-flow spectrophotometry, we observed a new transient intermediate (T201Speroxo) with a broad absorption band at λmax~ 650 nm (Figure 1 and Figure S1) and Δλmax~ 700 nm (Figure S2). This feature provides optical spectroscopic evidence for an oxygenated intermediate in toluene monooxygenase. A typical kinetic trace of the growth and decay of the transient intermediate is provided in the inset, together with the fit. The absorbance at 675 nm maximizes at ~ 40 ms after dioxygen is mixed at 4.0 ± 0.1 °C. Fitting the time-dependent absorption spectra to a ToMOHred→T201Speroxo→diiron(III) product model yielded kform = 85 ± 11 s−1 and kdecay = 2.9 ± 0.2 s−1. The intermediate is only observed in the presence of the regulatory protein (ToMOD), which is consistent with previous reports on oxygenated intermediates in BMMs such as Hperoxo or Q in sMMOH,24 or ToMOHperoxo in wild type11 or I100W ToMOH.25

Figure 1
UV-Vis spectrum of the reaction of reduced ToMOH T201S in the presence of ToMOD mixed with dioxygen-saturated buffer. [ToMOH] = ~ 120 µM, [ToMOD] = 360 µM in 25 mM MOPS, pH 7.0 at 4.0 ± 0.1 °C. (Inset) The time-dependent ...

To investigate further the spectroscopic properties of this intermediate, we performed Mössbauer studies with 57Fe-enriched T201S ToMOH enzyme. We present the Mössbauer spectra of reduced diiron(II) in the presence of ToMOD (Figure 2A), a rapid freeze-quenched sample at 45 ms (Figure 2B), and the diiron(III) product (Figure 2C). The spectra corresponding to the diiron(II) starting material and diiron(III) product of T201S ToMOH were respectively fit to (i) two quadrupole doublets having the same δ = 1.32 mm/s, ΔEQ= 2.32 and 3.16 mm/s and (ii) δ = 0.50 mm/s, ΔEQ= 0.82. Within experimental error, these parameters are indistinguishable from those of wild type ToMOH. When T201S ToMOHred was allowed to react with dioxygen (Figure 2B), we observed that only 50% of the diiron(II) sites react, whereas the rest becomes slowly oxidized to a diiron(III) species, suggesting half-sites reactivity as in wild type ToMOH and the small subunit of ribonucleotide reductase.11,26

Figure 2
Mössbauer spectra of freeze-quenched samples from reaction of ToMOHredT201S:3ToMOD with O2. The spectra (vertical bars) are collected at 4.2 K in a 50-mT field parallel to the γ-beam. The three spectra correspond to (A) the diiron(II) ...

Upon reaction of T201S ToMOHred with dioxygen in the presence of ToMOD, two distinctive transient intermediates accumulate (Figure 2B). One of the intermediates displays Mössbauer parameters very similar to those of the diiron(III) intermediate in wild type ToMOH, with δ = 0.55 mm/s and ΔEQ = 0.70 mm/s. This intermediate, ToMOHperoxo, accounts for approximately 40% of total iron at 45 ms and fully decays by ~ 100 sec (Figure 2C), exhibiting kinetic behavior similar to that of ToMOHperoxo in the wild type enzyme. As in the wild type, ToMOHperoxo generated in T201S also has no optical band in the visible range.

The other intermediate displays Mössbauer parameters identical to those of Hperoxo in sMMOH with δ = 0.67 mm/s and ΔEQ= 1.51 mm/s, implying that structure of T201Speroxo is similar to the structure of Hperoxo in sMMOH, but different from that of ToMOHperoxo. This intermediate accounts for 10% of total iron in the Mössbauer spectrum of the sample (Figure 2B), which allows us to estimate the molar extinction coefficient of T201Speroxo to be ε675 ~ 1500 cm−1 M−1. This value is within the range of those reported for peroxo-to-iron charge transfer bands in several diiron(III) peroxo enzyme intermediates14,15,17,18,27 as well as synthetic model complexes.28,29 Given these spectroscopic similarities, we assign this species as a peroxodiiron(III) intermediate. Moreover, the kinetic properties of the two discrete diiron(III) intermediates demonstrate that T201Speroxo is not a precursor of ToMOHperoxo, for which kform ~ 26 s−1 implying that T201S has an additional, alternative pathway of activating dioxygen.

In conclusion, we report the first observation of an oxygenated intermediate having an optical band in ToMOH, generated by a single, conservative mutation of the T201 residue. Although during dioxygen activation T201S generates T201Speroxo and ToMOHperoxo, the former being similar to MMOHperoxo, it behaves in a manner similar to the wild type enzyme in steady state activity and single turnover experiments. It also has regiospecificity comparable to that of the wild type enzyme in toluene and o-xylene oxidations. Time-resolved optical and rapid freeze-quench Mössabuer experiments strongly support the assignment of the oxygenated intermediate in ToMOH, T201Speroxo, as a peroxodiiron(III) species. Although further studies are required to define the mechanism of dioxygen activation and the role of the T201 residue in ToMOH, our results raise the interesting possibility that this single amino acid perturbs the thermodynamics of dioxygen activation in carboxylate-bridged non-heme diiron enzymes.

Supplementary Material

1_si_001

Acknowledgment

This work was supported by grants GM32134 (to SJL) and GM 47295 (to BHH) from the National Institute of General Medical Sciences. RKB acknowledges fellowship support from the NIGMS (1 F32 GM084564-01). We thank Prof. J. Stubbe for use of her freeze-quench apparatus and Dr. L. J. Murray for helpful comments on the manuscript.

Footnotes

Supporting Information Available: Experimental details. This material is available free of charge via the Internet at http://pubs.acs.org/.

REFERENCES

1. Merkx M, Kopp DA, Sazinsky MH, Blazyk JL, Müller J, Lippard SJ. Angew. Chem. Int. Ed. 2001;40:2782–2807. [PubMed]
2. Wallar BJ, Lipscomb JD. Chem. Rev. 1996;96:2625–2657. [PubMed]
3. Stenkamp RE. Chem. Rev. 1994;94:715–726.
4. Stubbe J, Nocera DG, Yee CS, Chang MCY. Chem. Rev. 2003;103:2167–2202. [PubMed]
5. Rosenzweig AC, Frederick CA, Lippard SJ, Nordlund P. Nature. 1993;366:537–543. [PubMed]
6. Holmes MA, Le Trong I, Turley S, Sieker LC, Stenkamp RE. J. Mol. Biol. 1991;218:583–593. [PubMed]
7. Nordlund P, Eklund H. J. Mol. Biol. 1993;232:123–164. [PubMed]
8. Abbreviations: RNR-R2, E. Coli ribonucleotide reductase R2 subunit; ToMO, toluene/o-xylene monooxygenase; MMOH, methane monooxygenase hydroxylase; BMM, bacterial multicomponent monooxygenase; Δ9D, Δ9-desaturase; T4moH, toluene 4-monooxygenase.
9. Baldwin J, Voegtli WC, Khidekel N, Moënne-Loccoz P, Krebs C, Pereira AS, Ley BA, Huynh BH, Loehr TM, Riggs-Gelasco PJ, Rosenzweig AC, Bollinger JM., Jr J. Am. Chem. Soc. 2001;123:7017–7030. [PubMed]
10. Murray LJ, Garcia-Serres R, McCormick MS, Davydov R, Naik SG, Kim S-H, Hoffman BM, Huynh BH, Lippard SJ. Biochemistry. 2007;46:14795–14809. [PMC free article] [PubMed]
11. Murray LJ, Naik SG, Ortillo DO, García-Serres R, Lee JK, Huynh BH, Lippard SJ. J. Am. Chem. Soc. 2007;129:14500–14510. [PMC free article] [PubMed]
12. Coufal DE, Blazyk JL, Whittington DA, Wu WW, Rosenzweig AC, Lippard SJ. Eur. J. Biochem. 2000;267:2174–2185. [PubMed]
13. Sazinsky MH, Bard J, Di Donato A, Lippard SJ. J. Biol. Chem. 2004;279:30600–30610. [PubMed]
14. Liu KE, Valentine AM, Qiu D, Edmondson DE, Appelman EH, Spiro TG, Lippard SJ. J. Am. Chem. Soc. 1995;117:4997–4998.
15. Lee S-K, Lipscomb JD. Biochemistry. 1999;38:4423–4432. [PubMed]
16. Liu KE, Wang D, Huynh BH, Edmondson DE, Salifoglou A, Lippard SJ. J. Am. Chem. Soc. 1994;116:7465–7466.
17. Broadwater JA, Ai J, Loehr TM, Sanders-Loehr J, Fox BG. Biochemistry. 1998;37:14664–14671. [PubMed]
18. Bollinger JM, Jr, Krebs C, Vicol A, Chen S, Ley BA, Edmondson DE, Huynh BH. J. Am. Chem. Soc. 1998;120:1094–1095.
19. Moënne-Loccoz P, Baldwin J, Ley BA, Loehr TM, Bollinger JM., Jr Biochemistry. 1998;37:14659–14663. [PubMed]
20. Bailey LJ, McCoy JG, Phillips J, George N, Fox BG. Proc. Natl. Acad. Sci. 2008;105:19194–19198. [PubMed]
21. Cafaro V, Scognamiglio R, Viggiani A, Izzo V, Passaro I, Notomista E, Dal Piaz F, Amoresano A, Casbarra A, Pucci P, Di Donato A. Eur. J. Biochem. 2002;269:5689–5699. [PubMed]
22. Tinberg CE, Song WJ, Izzo V, Lippard SJ. Manuscript in preparation.
23. Cafaro V, Notomista E, Capasso P, Di Donato A. Appl. Environ. Microbiol. 2005;71:4736–4743. [PMC free article] [PubMed]
24. Liu Y, Nesheim JC, Lee S-K, Lipscomb JD. J. Biol. Chem. 1995;270:24662–24665. [PubMed]
25. Murray LJ, García-Serres R, Naik S, Huynh BH, Lippard SJ. J. Am. Chem. Soc. 2006;128:7458–7459. [PMC free article] [PubMed]
26. Sjöberg B-M, Karlsson M, Jörnvall H. J. Biol. Chem. 1987;262:9736–9743. [PubMed]
27. Pereira AS, Small W, Krebs C, Tavares P, Edmondson DE, Theil EC, Huynh BH. Biochemistry. 1998;37:9871–9876. [PubMed]
28. Kim K, Lippard SJ. J. Am. Chem. Soc. 1996;118:4914–4915.
29. Yamashita M, Furutachi H, Tosha T, Fujinami S, Saito W, Maeda Y, Takahashi K, Tanaka K, Kitagawa T, Suzuki M. J. Am. Chem. Soc. 2007;129:2–3. [PubMed]