Effects of Dipole–Dipole Interactions on Microwave Progressive Power Saturation of Radicals in Proteins

Galli, C.; Innes, Jennifer B.; Hirsh, Donald J.; Brudvig, Gary W.

doi:10.1006/jmrb.1996.0044

Cited by 36 publications

(60 citation statements)

References 6 publications

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“…We derived fitting parameters of 1.0 milliwatts for P 1/2 and 0.77 for b, suggesting strong dipolar coupling between BH3 and high spin ferric heme (33). In contrast, the 4-amino-BH3 radical showed signs of partial saturation already at 10 microwatts (Fig.…”

Section: Resultsmentioning

confidence: 94%

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Single-turnover of Nitric-oxide Synthase in the Presence of 4-Amino-tetrahydrobiopterin

Sørlie

Gorren

Marchal

et al. 2003

Journal of Biological Chemistry

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Tetrahydrobiopterin (BH4) is an essential cofactor of nitric-oxide synthase (NOS) that serves as a one-electron donor to the oxyferrous⅐heme complex. 4-Aminotetrahydrobiopterin (4-amino-BH4) is a potent inhibitor of NO synthesis, although it mimics all allosteric and structural effects of BH4 and exhibits comparable redox properties. We studied the reaction of reduced endothelial NOS oxygenase domain with O 2 in the presence of 4-amino-BH4 at ؊30°C by optical and electron paramagnetic resonance (EPR) spectroscopy. With Arg as the substrate, we observed a trihydropteridine radical with a corresponding heme species that was oxyferrous, with a Soret maximum at 428 nm and no EPR signal. With N G -hydroxy-L-arginine (NHA) no pterin radical appeared, whereas an axial ferrous heme⅐NO complex was formed. The corresponding optical spectra, with Soret bands at 417/423 nm, suggest that the proximal sulfur ligand is protonated. Accordingly, 4-amino-BH4 serves as a one-electron donor to Fe(II)⅐O 2 with both Arg and NHA, but the reaction cycle cannot be completed with either substrate. We propose that protonation of Fe(II)O 2 ؊ is inhibited in the presence of 4-amino-BH4. With Arg, dissociation of O 2 ؊ and binding of O 2 yields Fe(II)⅐O 2 and a pteridine radical; with NHA, reaction of the substrate with heme-bound O 2 ؊ eventually yields Fe(II)⅐NO and reduced 4-amino-BH4. These results suggest that BH4 donates a proton to Fe(II)⅐O 2 ؊ during catalysis and that inhibition by 4-amino-BH4 may be due to its inability to support this essential protonation step.

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Section: Resultsmentioning

confidence: 94%

“…The low value of P 1/2 indicates that the majority of the radical is magnetically isolated, resulting in slow relaxation. However, the extremely low b value is suggestive of heterogeneity, with a minor part experiencing dipolar coupling with the heme (33).…”

Section: Resultsmentioning

confidence: 99%

Single-turnover of Nitric-oxide Synthase in the Presence of 4-Amino-tetrahydrobiopterin

Sørlie

Gorren

Marchal

et al. 2003

Journal of Biological Chemistry

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“…The power saturation data in Figs. 4 and 5 were (also) analyzed using nonlinear least-squares regression onto the data of a semiempirical equation 7 (see also Table II) (29,30) using the program Kaleidograph.…”

Section: Methodsmentioning

confidence: 99%

“…Thus, it is physically impossible for an isolated spin system to be characterized by b Ͻ 1, and such a scenario is therefore diagnostic of a dipolar interaction (30).…”

Section: Epr Power Saturation Behavior Of the S-3 Center In The Air-omentioning

confidence: 99%

Electron Paramagnetic Resonance Studies of Succinate:Ubiquinone Oxidoreductase from Paracoccus denitrificans

Waldeck¹,

Stowell²,

Lee³

et al. 1997

Journal of Biological Chemistry

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Electron paramagnetic resonance (EPR) studies of succinate:ubiquinone oxidoreductase (SQR) from Paracoccus denitrificans have been undertaken in the purified and membrane-bound states. Spectroscopic "signatures" accounting for the three iron-sulfur clusters (2Fe-2S, 3Fe-4S, and 4Fe-4S), cytochrome b, flavin, and protein-bound ubisemiquinone radicals have been obtained in air-oxidized, succinate-reduced, and dithionite-reduced preparations at 4 -10 K. Spectra obtained at 170 K in the presence of excess succinate showed a signal typical of that of a flavin radical, but superimposed with another signal. The superimposed signal originated from two bound ubisemiquinones, as shown by spectral simulations. Power saturation measurements performed on the air-oxidized enzyme provided evidence for a weak magnetic dipolar interaction operating between the oxidized 3Fe-4S cluster and the oxidized cytochrome b. Power saturation experiments performed on the succinate-and dithionite-reduced forms of the enzyme demonstrated that the 4Fe-4S cluster is coupled weakly to both the 2Fe-2S and the 3Fe-4S clusters. Quantitative interpretation of these power saturation experiments has been achieved through redox calculations. They revealed that a spin-spin interaction between the reduced 3Fe-4S cluster and the cytochrome b (oxidized) may also exist. These findings form the first direct EPR evidence for a close proximity ( 2 nm) of the high potential 3Fe-4S cluster, situated in the succinate dehydrogenase part of the enzyme, and the low potential, low spin b-heme in the membrane anchor of the enzyme.Succinate:ubiquinone oxidoreductase, SQR, 1 is the only membrane-bound enzyme in the tricarboxylic acid cycle. As "complex II," it performs the two-electron oxidation of succinate to produce fumarate, while transferring the electrons to quinone (Q) to yield quinol (QH 2 ). The reverse process is mediated by quinol:fumarate oxidoreductases (QFR), which occur in anaerobic and some facultative organisms. The two enzymes are related and are capable of catalyzing their respective reverse reactions under suitable conditions (1, 2).SQR contains three or four polypeptides depending on the organism. The largest subunit, a flavoprotein, contains the dicarboxylate binding site and one flavin moiety (FAD); the latter is covalently bound in most cases. The iron-sulfur protein is intermediate in size and contains three iron-sulfur clusters of type 2Fe-2S, 4Fe-4S, and 3Fe-4S, often referred to as S-1, S-2, and S-3, respectively, in the case of SQR. These two hydrophilic subunits protrude into the cytosol (prokaryotic enzyme) or the mitochondrial matrix (eukaryotic enzyme), and together catalyze the succinate dehydrogenase activity of the enzyme. They are anchored to the membrane by one or two hydrophobic quinone polypeptides (QP), which may contain zero, one, or two b-heme(s). These anchoring subunits confer reactivity with the bound Qs; for SQR from bovine heart (3, 4) and a variety of higher plants (5), the existence of two Q sites has been established. S...

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“…Because the dipole-dipole interaction is orientation dependent, radicals with different orientations of the interspin vector in the magnetic field will have different values of k 1y and, therefore, different values of k 1 (equation (3)). The result is that there is no single value of P 1/2 that can characterize the power at half saturation (equation (5)) but rather a range of values whose extent depends on the strength of the dipole-dipole interaction 29 . The effect is to ''stretch'' the P 1/2 curve along the x axis (microwave power).…”

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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Effects of Dipole–Dipole Interactions on Microwave Progressive Power Saturation of Radicals in Proteins

Cited by 36 publications

References 6 publications

Single-turnover of Nitric-oxide Synthase in the Presence of 4-Amino-tetrahydrobiopterin

Single-turnover of Nitric-oxide Synthase in the Presence of 4-Amino-tetrahydrobiopterin

Electron Paramagnetic Resonance Studies of Succinate:Ubiquinone Oxidoreductase from Paracoccus denitrificans

Measuring distances in proteins by saturation-recovery EPR

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