### Abstract

The stable tyrosine radical of ribonucleotide reductase (RNR) from Escherichia coli, Tyr 122 of the B2 subunit, exhibits single-exponential spin-lattice relaxation kinetics for T ≤ 16 K and nonexponential spin-lattice relaxation kinetics at higher temperatures. Saturation-recovery transients of the tyrosine radical are analyzed using a model developed to treat the interaction of two paramagnets in a rigid lattice at a fixed distance apart but with a randon orientation in the static magnetic field. The model describes the spin-lattice relaxation of a radical in proximity to another paramagnetic site in terms of two isotropic or "scalar" rate constants and an orientation-dependent rate constant. The scalar rate constants arise from (1) intrinsic relaxation processes of the radical which exist in the absence of the other paramagnetic site and (2) a scalar-exchange-induced relaxation process arising from orbital overlap between the two paramagnetic sites. The orientation-dependent rate constant arises from a dipole-dipole-induced relaxation process. From simulations of the higher temperature saturation-recovery transients, we conclude that their nonexponential character arises from a dipole-dipole interaction with the diferric center of RNR. The tyrosine radical generated by UV photolysis of L-tyrosine in a borate glass is used as a model for the intrinsic spin-lattice relaxation rate of the tyrosine radical of ribonucleotide reductase. Comparison of the scalar rate constants derived from simulations of the saturation-recovery transients of the tyrosine radical of RNR with the single-exponential rate constants of the model tyrosine radical indicates scalar exchange is also a source of relaxation enhancement of the tyrosine radical of RNR at higher temperatures. We present a new method for determining the exchange coupling of the diferric center based on the temperature dependence of the scalar-exchange and dipolar rate constants. The Fe(III)-Fe(III) exchange coupling is estimated to be -94 ± 7 cm^{-1}. We also estimate an exchange coupling of |0.0047 ± 0.0003 cm^{-1}+| between the diferric center and the tyrosine radical on the basis of the relative contributions of scalar-exchange and dipolar interactions to the spin-lattice relaxation and the distance between the two sites. The source of Line broadening of the EPR signal of the tyrosine radical of RNR at temperatures greater than 75 K is discussed as well.

Original language | English |
---|---|

Pages (from-to) | 7475-7481 |

Number of pages | 7 |

Journal | Journal of the American Chemical Society |

Volume | 114 |

Issue number | 19 |

Publication status | Published - 1992 |

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### ASJC Scopus subject areas

- Chemistry(all)

### Cite this

*Journal of the American Chemical Society*,

*114*(19), 7475-7481.

**Using saturation-recovery EPR to measure exchange couplings in proteins : Application to ribonucleotide reductase.** / Hirsh, Donald J.; Beck, Warren F.; Brudvig, Gary W; Que, Lawrence; Brudvig, Gary W.

Research output: Contribution to journal › Article

*Journal of the American Chemical Society*, vol. 114, no. 19, pp. 7475-7481.

}

TY - JOUR

T1 - Using saturation-recovery EPR to measure exchange couplings in proteins

T2 - Application to ribonucleotide reductase

AU - Hirsh, Donald J.

AU - Beck, Warren F.

AU - Brudvig, Gary W

AU - Que, Lawrence

AU - Brudvig, Gary W.

PY - 1992

Y1 - 1992

N2 - The stable tyrosine radical of ribonucleotide reductase (RNR) from Escherichia coli, Tyr 122 of the B2 subunit, exhibits single-exponential spin-lattice relaxation kinetics for T ≤ 16 K and nonexponential spin-lattice relaxation kinetics at higher temperatures. Saturation-recovery transients of the tyrosine radical are analyzed using a model developed to treat the interaction of two paramagnets in a rigid lattice at a fixed distance apart but with a randon orientation in the static magnetic field. The model describes the spin-lattice relaxation of a radical in proximity to another paramagnetic site in terms of two isotropic or "scalar" rate constants and an orientation-dependent rate constant. The scalar rate constants arise from (1) intrinsic relaxation processes of the radical which exist in the absence of the other paramagnetic site and (2) a scalar-exchange-induced relaxation process arising from orbital overlap between the two paramagnetic sites. The orientation-dependent rate constant arises from a dipole-dipole-induced relaxation process. From simulations of the higher temperature saturation-recovery transients, we conclude that their nonexponential character arises from a dipole-dipole interaction with the diferric center of RNR. The tyrosine radical generated by UV photolysis of L-tyrosine in a borate glass is used as a model for the intrinsic spin-lattice relaxation rate of the tyrosine radical of ribonucleotide reductase. Comparison of the scalar rate constants derived from simulations of the saturation-recovery transients of the tyrosine radical of RNR with the single-exponential rate constants of the model tyrosine radical indicates scalar exchange is also a source of relaxation enhancement of the tyrosine radical of RNR at higher temperatures. We present a new method for determining the exchange coupling of the diferric center based on the temperature dependence of the scalar-exchange and dipolar rate constants. The Fe(III)-Fe(III) exchange coupling is estimated to be -94 ± 7 cm-1. We also estimate an exchange coupling of |0.0047 ± 0.0003 cm-1+| between the diferric center and the tyrosine radical on the basis of the relative contributions of scalar-exchange and dipolar interactions to the spin-lattice relaxation and the distance between the two sites. The source of Line broadening of the EPR signal of the tyrosine radical of RNR at temperatures greater than 75 K is discussed as well.

AB - The stable tyrosine radical of ribonucleotide reductase (RNR) from Escherichia coli, Tyr 122 of the B2 subunit, exhibits single-exponential spin-lattice relaxation kinetics for T ≤ 16 K and nonexponential spin-lattice relaxation kinetics at higher temperatures. Saturation-recovery transients of the tyrosine radical are analyzed using a model developed to treat the interaction of two paramagnets in a rigid lattice at a fixed distance apart but with a randon orientation in the static magnetic field. The model describes the spin-lattice relaxation of a radical in proximity to another paramagnetic site in terms of two isotropic or "scalar" rate constants and an orientation-dependent rate constant. The scalar rate constants arise from (1) intrinsic relaxation processes of the radical which exist in the absence of the other paramagnetic site and (2) a scalar-exchange-induced relaxation process arising from orbital overlap between the two paramagnetic sites. The orientation-dependent rate constant arises from a dipole-dipole-induced relaxation process. From simulations of the higher temperature saturation-recovery transients, we conclude that their nonexponential character arises from a dipole-dipole interaction with the diferric center of RNR. The tyrosine radical generated by UV photolysis of L-tyrosine in a borate glass is used as a model for the intrinsic spin-lattice relaxation rate of the tyrosine radical of ribonucleotide reductase. Comparison of the scalar rate constants derived from simulations of the saturation-recovery transients of the tyrosine radical of RNR with the single-exponential rate constants of the model tyrosine radical indicates scalar exchange is also a source of relaxation enhancement of the tyrosine radical of RNR at higher temperatures. We present a new method for determining the exchange coupling of the diferric center based on the temperature dependence of the scalar-exchange and dipolar rate constants. The Fe(III)-Fe(III) exchange coupling is estimated to be -94 ± 7 cm-1. We also estimate an exchange coupling of |0.0047 ± 0.0003 cm-1+| between the diferric center and the tyrosine radical on the basis of the relative contributions of scalar-exchange and dipolar interactions to the spin-lattice relaxation and the distance between the two sites. The source of Line broadening of the EPR signal of the tyrosine radical of RNR at temperatures greater than 75 K is discussed as well.

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M3 - Article

VL - 114

SP - 7475

EP - 7481

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 19

ER -