Kinetics of transient radicals in Escherichia coli ribonucleotide reductase : Formation of a new tyrosyl radical in mutant protein R2
Katterle, Bettina ; Sahlin, Margareta ; Schmidt, Peter P. ; Pötsch, Stephan ; Logan, Derek T. LU
; Gräslund, Astrid
and Sjöberg, Britt-Marie
(1997)
In Journal of Biological Chemistry
272(16).
p.10414-10421
- Abstract
Reconstitution of the tyrosyl radical in ribonucleotide reductase protein R2 requires oxidation of a diferrous site by oxygen. The reaction involves one externally supplied electron in addition to the three electrons provided by oxidation of the Tyr-122 side chain and formation of the μ-oxo- bridged diferric site. Reconstitution of R2 protein Y122F, lacking the internal pathway involving Tyr-122, earlier identified two radical intermediates at Trp-107 and Trp-111 in the vicinity of the di-iron site, suggesting a novel internal transfer pathway (Sahlin, M., Lassmann, G., Potsch, S., Sjoberg, B.-M., and Graslund, A. (1995) J. Biol. Chem. 270, 12361-12372). Here, we report the construction of the double mutant W107Y/Y122F and its... (More)
Reconstitution of the tyrosyl radical in ribonucleotide reductase protein R2 requires oxidation of a diferrous site by oxygen. The reaction involves one externally supplied electron in addition to the three electrons provided by oxidation of the Tyr-122 side chain and formation of the μ-oxo- bridged diferric site. Reconstitution of R2 protein Y122F, lacking the internal pathway involving Tyr-122, earlier identified two radical intermediates at Trp-107 and Trp-111 in the vicinity of the di-iron site, suggesting a novel internal transfer pathway (Sahlin, M., Lassmann, G., Potsch, S., Sjoberg, B.-M., and Graslund, A. (1995) J. Biol. Chem. 270, 12361-12372). Here, we report the construction of the double mutant W107Y/Y122F and its three-dimensional structure and demonstrate that the tyrosine Tyr-107 can harbor a transient, neutral radical (Tyr-107(·)). The Tyr-107(·) signal exhibits the hyperfine structure of a quintet with coupling constants of 1.3 mT for one 0-methylene proton and 0.75 mT for each of the 3 and 5 hydrogens of the phenyl ring. Rapid freeze quench kinetics of EPR-visible intermediates reveal a preferred radical transfer pathway via Trp-111, Glu-204, and Fe-2, followed by a proton coupled electron transfer through the π-interaction of the aromatic rings of Trp-(Tyr-)107 and Trp- 111. The kinetic pattern observed in W107Y/Y122F is considerably changed as compared with Y122F: the Trp-111(·) EPR signal has vanished, and the Tyr- 107 has the same formation rate as does Trp-111(·) in Y122F. According to the proposed consecutive reaction, Trp-111(·) becomes very short lived and is no longer detectable because of the faster formation of Tyr-107(·). We conclude that the phenyl rings of Trp-111 and Tyr-107 form a better stacking complex so that the proton-coupled electron transfer is facilitated compared with the single mutant. Comparison with the formation kinetics of the stable tyrosyl radical in wild type R2 suggests that these protein-linked radicals are substitutes for the missing Tyr-122. However, in contrast to Tyr-122 these radicals lack a direct connection to the radical transfer pathway utilized during catalysis.
(Less)
- author
- Katterle, Bettina
; Sahlin, Margareta
; Schmidt, Peter P.
; Pötsch, Stephan
; Logan, Derek T.
LU
; Gräslund, Astrid
and Sjöberg, Britt-Marie
- publishing date
- 1997-04-18
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Journal of Biological Chemistry
- volume
- 272
- issue
- 16
- pages
- 8 pages
- publisher
- American Society for Biochemistry and Molecular Biology
- external identifiers
-
- pmid:9099682
- scopus:0030968307
- ISSN
- 0021-9258
- DOI
- 10.1074/jbc.272.16.10414
- language
- English
- LU publication?
- no
- id
- 4406c1ca-4285-4742-851b-276beacdbccd
- date added to LUP
- 2022-04-25 11:26:31
- date last changed
- 2024-01-03 10:38:05
@article{4406c1ca-4285-4742-851b-276beacdbccd,
abstract = {{<p>Reconstitution of the tyrosyl radical in ribonucleotide reductase protein R2 requires oxidation of a diferrous site by oxygen. The reaction involves one externally supplied electron in addition to the three electrons provided by oxidation of the Tyr-122 side chain and formation of the μ-oxo- bridged diferric site. Reconstitution of R2 protein Y122F, lacking the internal pathway involving Tyr-122, earlier identified two radical intermediates at Trp-107 and Trp-111 in the vicinity of the di-iron site, suggesting a novel internal transfer pathway (Sahlin, M., Lassmann, G., Potsch, S., Sjoberg, B.-M., and Graslund, A. (1995) J. Biol. Chem. 270, 12361-12372). Here, we report the construction of the double mutant W107Y/Y122F and its three-dimensional structure and demonstrate that the tyrosine Tyr-107 can harbor a transient, neutral radical (Tyr-107(·)). The Tyr-107(·) signal exhibits the hyperfine structure of a quintet with coupling constants of 1.3 mT for one 0-methylene proton and 0.75 mT for each of the 3 and 5 hydrogens of the phenyl ring. Rapid freeze quench kinetics of EPR-visible intermediates reveal a preferred radical transfer pathway via Trp-111, Glu-204, and Fe-2, followed by a proton coupled electron transfer through the π-interaction of the aromatic rings of Trp-(Tyr-)107 and Trp- 111. The kinetic pattern observed in W107Y/Y122F is considerably changed as compared with Y122F: the Trp-111(·) EPR signal has vanished, and the Tyr- 107 has the same formation rate as does Trp-111(·) in Y122F. According to the proposed consecutive reaction, Trp-111(·) becomes very short lived and is no longer detectable because of the faster formation of Tyr-107(·). We conclude that the phenyl rings of Trp-111 and Tyr-107 form a better stacking complex so that the proton-coupled electron transfer is facilitated compared with the single mutant. Comparison with the formation kinetics of the stable tyrosyl radical in wild type R2 suggests that these protein-linked radicals are substitutes for the missing Tyr-122. However, in contrast to Tyr-122 these radicals lack a direct connection to the radical transfer pathway utilized during catalysis.</p>}},
author = {{Katterle, Bettina and Sahlin, Margareta and Schmidt, Peter P. and Pötsch, Stephan and Logan, Derek T. and Gräslund, Astrid and Sjöberg, Britt-Marie}},
issn = {{0021-9258}},
language = {{eng}},
month = {{04}},
number = {{16}},
pages = {{10414--10421}},
publisher = {{American Society for Biochemistry and Molecular Biology}},
series = {{Journal of Biological Chemistry}},
title = {{Kinetics of transient radicals in <i>Escherichia coli</i> ribonucleotide reductase : Formation of a new tyrosyl radical in mutant protein R2}},
url = {{http://dx.doi.org/10.1074/jbc.272.16.10414}},
doi = {{10.1074/jbc.272.16.10414}},
volume = {{272}},
year = {{1997}},
}