Distance between the electron paramagnetic resonance-visible copper and cytochrome a in bovine heart cytochrome oxidase

Goodman, G; Leigh, Jr Js

doi:10.1021/bi00330a028

Cited by 51 publications

(33 citation statements)

References 25 publications

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“…The superimposition, however, of the g = 2.24 line of heme a in COX made the lines from Cu in this region more difficult to interpret. In addition, the lines at g, of the EPR-detectable Cu site relax about an order of magnitude faster than g , or g, (Goodman and Leigh, 1985). Thus, the lines in the g, rcgion are broader, and the hyperfine coupling is poorly resolved.…”

Section: Discussionmentioning

confidence: 97%

A comparative EPR investigation of the multicopper proteins nitrous‐oxide reductase and cytochrome c oxidase

Antholine

Kastrau

Steffens

et al. 1992

European Journal of Biochemistry

185

170

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The multicopper proteins, nitrous-oxide reductase (N20R) and cytochrome c oxidase (COX), were investigated by EPR spectroscopy at microwave frequencies 2.4 -35 GHz. Our results support a Cu-Cu interaction in COX and N20R. At least 10 lines in the 2.7-GHz, 12 lines in the 4.6-GHz and 14 lines in the 9.2 GHz spectra were resolved for N20R. Eight copper lines at 2.7 GHz, about ninc lines at 4.6 GHz and about six lines at 9.2 G H L were resolved for COX. Simulations of the EPR spectra were consistent with most of the resonances of the multiline spectra, including regions in the center of the spectra where overlap of the three seven-line patterns is proposed. These simulations indicated that Cu-Cu interaction, in a mixed-valence [Cu(l.5). . .Cu(l.5)], S = site is consistent with, if not proof of, the unusual spectral features observed for N 2 0 R and COX.Nitrous-oxide reductase (N20R) is the terminal electron acceptor in a respiratory chain converting N 2 0 to N2 in denitrifying bacteria: N 2 0 + 2H' + 2e-N2 + H 2 0 .

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Section: Discussionmentioning

confidence: 97%

A comparative EPR investigation of the multicopper proteins nitrous‐oxide reductase and cytochrome c oxidase

Antholine

Kastrau

Steffens

et al. 1992

European Journal of Biochemistry

185

170

View full text Add to dashboard Cite

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“…The second paramagnet is thought to be the manganese cluster that is involved in charge storage and probably acts as the active site for oxygen evolution (5,6). The dipolar spin-relaxation mechanism has been thoroughly studied (7)(8)(9). The strength of the dipolar interaction is determined by the distance between the two spins and the angle between the interspin vector and the applied magnetic field.…”

mentioning

confidence: 99%

Angular orientation of the stable tyrosyl radical within photosystem II by high-field 245-GHz electron paramagnetic resonance.

Brunel

Brill

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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The 4 K 245-GHz/8.7-T electron paramagnetic resonance spectrum of the stable tyrosyl radical in photosystem I, known as TyrD, has been measured. Illumination at 200 K enhances the signal Intensity of TyrD" by a factor of >40 compared to the signal obtained from darkadapted samples. This signal ehncement and the unusual ine shape of the TyrD' resonance result from the magnetic dipolar coupling of the radical to the manganese duster involved in oxygen evolution. The relative alar orientation of the m _ cluster with respect to TyrD-has been determined from line-shape analysis. The resonance arising from TyrD' in Tris-washed nsfree photosystem U sample is also distorted. This effect probably originates from the influence of the nonheme iron on the spin aon of the tyrosyl radical.The relative angular orientation of the nonheme iron has also been determined. Oriented samples were used to determine the angular orientation of TyrD with respect to the membrane plane. Combining angular data with pubilshed distances, we have constructed a three-dimensional picture of the relative positions of TyrD', the manganese cluster, and the noneme iron. The data suggest a more symmetrical placement of the manganese relative to TyrD-and TyrZ, the tyrosine involved in eleco transfer, than is usually assumed in current models of photosystem H.At low temperatures, the electron paramagnetic resonance (EPR) signal of the stable tyrosyl radical, known as TyrD- (1, 2), from plant photosystem II (PSIT) is known to be "relaxation enhanced" when the sample is illuminated at 200 K (3, 4). This enhancement has been attributed to the presence of a second paramagnetic species that is capable of increasing the relaxation rates of the tyrosyl radical through a dipolar interaction. The second paramagnet is thought to be the manganese cluster that is involved in charge storage and probably acts as the active site for oxygen evolution (5, 6). The dipolar spin-relaxation mechanism has been thoroughly studied (7-9). The strength of the dipolar interaction is determined by the distance between the two spins and the angle between the interspin vector and the applied magnetic field. Previous work on PSII has concentrated on the determination of the interspin distance (10-13). No attempts have been made to obtain the angular orientation of the two spins.At magnetic field strengths used in conventional EPR spectroscopy, the spectrum of the tyrosyl radical is dominated by the hyperfine interactions between the electron spin and its neighboring protons. The inhomogeneous line width of the spectrum is determined by the anisotropic parts of these hyperfine interactions and the g anisotropy, each of which has its own particular orientation dependence relative to the molecular frame. A good approximation is that all possible orientations ofthe radical with respect to the applied magnetic field contribute to a given field position on the TyrD spectrum. Thus, the determination ofthe orientation of an external relaxer is difficult. However, at magnetic fields m...

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“…There are two mechanisms by which the metal center can enhance the radical's spin-lattice relaxation rate [15][16][17][18][19][20] . One mechanism is the dipole-dipole interaction, a through-space mechanism that only requires proximity between the radical and the metal center.…”

Section: Dipole-dipole-and Exchange-induced Relaxation Enhancementmentioning

confidence: 99%

“…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: Samples For the Measurement Of K 1imentioning

confidence: 99%

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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Distance between the electron paramagnetic resonance-visible copper and cytochrome a in bovine heart cytochrome oxidase

Cited by 51 publications

References 25 publications

A comparative EPR investigation of the multicopper proteins nitrous‐oxide reductase and cytochrome c oxidase

A comparative EPR investigation of the multicopper proteins nitrous‐oxide reductase and cytochrome c oxidase

Angular orientation of the stable tyrosyl radical within photosystem II by high-field 245-GHz electron paramagnetic resonance.

Measuring distances in proteins by saturation-recovery EPR

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