Using saturation-recovery EPR to measure distances in proteins: applications to photosystem II

Hirsh, Donald J.; Beck, Warren F.; Innes, Jennifer B.; Brudvig, Gary W.

doi:10.1021/bi00117a033

Cited by 82 publications

(156 citation statements)

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“…Furthermore, magnetic interactions depend on temperature when one of the species of the interacting pair has a relaxation rate (T 1 ) faster than the other one. This may produce an enhancement of the relaxation rate of the slowly relaxing paramagnetic center and/or a temperature dependence of the splitting of the resonance lines [30,31,32,33]. Figure 3A shows that the Mo(V) resonances are fully split at temperatures below 50 K, partially collapsed in the 55-80 K range, and fully collapsed at 100 K (no linewidth changes were observed above 100 K and below 50 K).…”

Section: Analysis Of the Magnetic Interactionsmentioning

confidence: 99%

Incorporation of either molybdenum or tungsten into formate dehydrogenase from Desulfovibrio alaskensis NCIMB 13491; EPR assignment of the proximal iron-sulfur cluster to the pterin cofactor in formate dehydrogenases from sulfate-reducing bacteria

et al. 2003

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We report the characterization of the molecular properties and EPR studies of a new formate dehydrogenase (FDH) from the sulfate-reducing organism Desulfovibrio alaskensis NCIMB 13491. FDHs are enzymes that catalyze the two-electron oxidation of formate to carbon dioxide in several aerobic and anaerobic organisms. D. alaskensis FDH is a heterodimeric protein with a molecular weight of 126±2 kDa composed of two subunits, a=93±3 kDa and b=32±2 kDa, which contains 6±1 Fe/molecule, 0.4±0.1 Mo/molecule, 0.3±0.1 W/molecule, and 1.3±0.1 guanine monophosphate nucleotides. The UV-vis absorption spectrum of D. alaskensis FDH is typical of an iron-sulfur protein with a broad band around 400 nm. Variable-temperature EPR studies performed on reduced samples of D. alaskensis FDH showed the presence of signals associated with the different paramagnetic centers of D. alaskensis FDH. Three rhombic signals having g-values and relaxation behavior characteristic of [4Fe-4S] clusters were observed in the 5-40 K temperature range. Two EPR signals with all the g-values less than two, which accounted for less than 0.1 spin/protein, typical of mononuclear Mo(V) and W(V), respectively, were observed. The signal associated with the W(V) ion has a larger deviation from the free electron g-value, as expected for tungsten in a d 1 configuration, albeit with an unusual relaxation behavior. The EPR parameters of the Mo(V) signal are within the range of values typically found for the slow-type signal observed in several Mo-containing proteins belonging to the xanthine oxidase family of enzymes. Mo(V) resonances are split at temperatures below 50 K by magnetic coupling with one of the Fe/S clusters. The analysis of the inter-center magnetic interaction allowed us to assign the EPR-distinguishable iron-sulfur clusters with those seen in the crystal structure of a homologous enzyme.

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Section: Analysis Of the Magnetic Interactionsmentioning

confidence: 99%

Incorporation of either molybdenum or tungsten into formate dehydrogenase from Desulfovibrio alaskensis NCIMB 13491; EPR assignment of the proximal iron-sulfur cluster to the pterin cofactor in formate dehydrogenases from sulfate-reducing bacteria

et al. 2003

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“…To evaluate equation (7) and model the actual recovery, we must have an explicit equation for k 1y . For the situation most likely to be encountered by the investigator, that is, a spectrometer operating at X-band (9 GHz) or higher, a metal center containing first row transition metals, and temperatures near or below that of liquid nitrogen, k 1y can be expressed as 3,19,20 Figure 5 | The anatomy of a CW saturationrecovery experiment. The defense pulse switches on before the high-power saturating pulse and stays on until a short time afterward (typically 1-5 ms) to protect the receiver electronics from microwave power reflected by the resonator.…”

Section: Vol2 No7 | 2007 | Nature Protocols Protocolmentioning

confidence: 99%

“…However, if none of these options is workable, it may be possible to use the spin-lattice relaxation rates of the same radical species in (frozen) solution as k 1i (ref. 3). (See Fig.…”

Section: Samples For the Measurement Of K 1imentioning

confidence: 99%

“…where sin y gives the appropriate weighting to each angle y and N is a normalization constant 3,35 . To evaluate equation (7) and model the actual recovery, we must have an explicit equation for k 1y .…”

Section: Samples For the Measurement Of K 1imentioning

confidence: 99%

“…1,2). Enzymes with multiple redox-active cofactors such as photosystem II (PSII) and ribonucleotide reductase (RNR) are obvious targets for this methodology 3,4 . Indeed, in these systems, saturation-recovery EPR may also be used to measure exchange couplings between redox partners 4 .…”

Section: Introductionmentioning

confidence: 99%

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Measuring distances in proteins by saturation-recovery EPR

Hirsh

Brudvig

2007

Nat Protoc

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We describe a protocol for detecting electron spin-spin interactions between a radical and a metal ion in a protein or protein complex by saturation-recovery electron paramagnetic resonance (EPR). This protocol can be used with a protein containing an endogenous metal center and either an endogenous or synthetic radical species. We suggest a two-step approach whereby dipole-dipole or exchange interactions are first detected by continuous-wave EPR experiments and then quantified by saturation-recovery EPR. The latter measurements make it possible to measure long distances to within a few Å ngstroms. The protocol for making distance measurements by saturation-recovery EPR will take approximately 6 days to complete. INTRODUCTIONThe techniques that first come to mind when thinking about protein structure determination are usually solution-state NMR or X-ray crystallography. However, a complete protein structure is not always available or necessary to answer some of the most interesting biophysical questions. Sometimes, the measurement of one distance (or a few) is sufficient to establish the plausibility of a mechanism or corroborate a proposed structure. If a protein or protein complex contains a metal-binding site and a radical species, the magnetic interaction between the unpaired electrons at these two sites can be detected and quantified by saturation-recovery EPR and the resulting data used to measure distances of B10-40 Å (refs. 1,2). Enzymes with multiple redox-active cofactors such as photosystem II (PSII) and ribonucleotide reductase (RNR) are obvious targets for this methodology 3,4 . Indeed, in these systems, saturation-recovery EPR may also be used to measure exchange couplings between redox partners 4 . However, saturation-recovery EPR can also be used to measure distances between an endogenous metal-binding site and a strategically placed spin label to yield structural information 5 .Long distances in macromolecules have been successfully measured using fluorescence resonance energy transfer [6][7][8] and by pulsed EPR methods such as double quantum coherence, ''2 + 1,'' and DEER sequences that use a pair of spin labels (radicals) [9][10][11] . Distance measurement by saturation recovery has an advantage over these other methods when the protein contains an endogenous paramagnetic metal center in that the protein has a built-in ''label.'' The other label, that is, the radical, may be either endogenous or introduced through spin-labeling methods [12][13][14] . Additionally, in the case where the paramagnetic metal center is itself of interest, the radical can serve as a reporter of its magnetic properties. For example, in our experiments with the B2 subunit of RNR, the stable tyrosine radical was used to determine the exchange coupling within the dinuclear iron center 4 .As pulsed EPR instruments are not yet as common as pulsed (Fourier-transform) NMR instruments, we recommend that the investigator first attempts to detect the presence of electron spinspin interactions using the more common continuous...

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Determination of High-Spin Iron(III)–Nitroxyl Distances in Spin-Labeled Porphyrins by Time-Domain EPR

Rakowsky

Zecevic

Eaton

1998

Journal of Magnetic Resonance

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Using saturation-recovery EPR to measure distances in proteins: applications to photosystem II

Cited by 82 publications

References 31 publications

Incorporation of either molybdenum or tungsten into formate dehydrogenase from Desulfovibrio alaskensis NCIMB 13491; EPR assignment of the proximal iron-sulfur cluster to the pterin cofactor in formate dehydrogenases from sulfate-reducing bacteria

Incorporation of either molybdenum or tungsten into formate dehydrogenase from Desulfovibrio alaskensis NCIMB 13491; EPR assignment of the proximal iron-sulfur cluster to the pterin cofactor in formate dehydrogenases from sulfate-reducing bacteria

Measuring distances in proteins by saturation-recovery EPR

Determination of High-Spin Iron(III)–Nitroxyl Distances in Spin-Labeled Porphyrins by Time-Domain EPR

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