Electron spin relaxation of iron-sulphur proteins studied by microwave power saturation

Rupp, Heinz; Rao, K. K.; Hall, D.O.; Cammack, Richard

doi:10.1016/0005-2795(78)90509-3

Cited by 194 publications

(139 citation statements)

References 32 publications

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“…3 compares the continuous wave microwave saturation behavior ofthe Co2+ ion in the free enzyme with that ofthe Co2+ ion in the enzyme-NAD+-pyrazole complex. The data are plotted in a form (17,18) that gives a graphical estimate of P1i2 according to the relationship S/VP cx (1 + p/p112)-b12, where S is the normalized signal amplitude; P is the microwave power incident on the cavity; P1i2, the microwave power at which the saturation factor (1 + yHl2TjT2)-1 is equal to 0.5, is determined by the intersection ofthe asymoptotic limits ofthe slopes; and b is a constant characteristic of the broadening mechanism and is a measure of the ratio of the spin-packet linewidth (proportional to 1/T2) to the total width of the unsaturated inhomogeneously broadened line envelope. The value of b is determined by the asymptotic limit of the slope at high values of P. For each complex of the enzyme, it is apparent from Fig.…”

Section: Resultsmentioning

confidence: 99%

Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

Makinen¹,

Yim²

1981

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

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The coordination environment of the catalytically active metal ion of horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) has been investigated by electron paramagnetic resonance (EPR) methods with use of the active-site-specific Co2+-reconstituted enzyme. The EPR absorption spectrum of the metal-substituted enzyme is characteristic of a rhombically distorted environment. The spectrum of the enzyme-NAD+ complex shows approximate axial symmetry of the metal ion site, indicating that binding of the coenzyme induces a structural alteration in the active-site region. This environment is not significantly altered further by binding of the competitive inhibitor pyrazole. To assign the coordination number of the active-site metal ion, the zero-field splitting was determined on the basis ofthe temperature dependence ofthe spin-lattice relaxation of the Co2+ ion. The zero-field splitting energies are '9 cm-' for the free Co2+-reconstituted enzyme and e46 and -47 cm-' for the enzyme-NAD+ and enzyme-NAD+-pyrazole complexes, respectively. On the basis of studies of structurally defined small molecule complexes, these values are compatible with a tetracoordinate metal ion in the active site of the free enzyme but a pentacoordinate metal ion in the binary enzyme-NAD+ complex and in the ternary enzyme-NAD+-inhibitor complex and, therefore, presumably also in the catalytically active ternary enzyme-NAD+-alcohol complex formed in the course of alcohol oxidation.The oxidation ofalcohol catalyzed by liver alcohol dehydrogenase (LADH; alcohol:NAD' oxidoreductase, EC 1.1.1.1) proceeds at the level of a ternary complex formed by the enzyme, the coenzyme NAD', and the substrate as the catalytically active intermediate of the reaction (1-3). In the free enzyme, the tetracoordinate Zn2+ ion in the active site is ligated by the sulfhydryl groups of cysteine-46 and cysteine-174, the NE atom of histidine-67, and a water molecule (4). The coordination number of the active-site metal ion is considerably less well assigned for reaction intermediates. The suggestion has been made on the basis of proton relaxation enhancement studies of enzyme-coenzyme-inhibitor complexes that substrates bind as outer sphere ligands only with no change in coordination number (5). Interpretations of the pH dependence of the kinetic parameters of the enzyme have invoked formation of both tetracoordinate (6, 7) and pentacoordinate (8, 9) intermediates as the catalytically active species. Difference Fourier syntheses of (alcohol) inhibitor substrate analogs bound to the active site of the enzyme-NAD' complex crystallized in the presence of 2-methyl-2,4-pentanediol suggest that the substrate displaces the metal-bound water molecule to form a tetracoordinate species only (10). However, the crystallizing reagent 2-methyl-2,4-pentanediol acts as substrate analog of the enzyme, resulting in a mixture of enzyme-NAD' and enzyme-NADH species in the crystal.* Under these conditions, addition of alcohol substrates may lead also to formation o...

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Section: Resultsmentioning

confidence: 99%

Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

Makinen¹,

Yim²

1981

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

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“…where A i is the amplitude of the ith-type free radical, C i is a coefficient for the actual content of the ith-type free radical in the sample, P 50(i) is the half-saturation microwave power, b ii is the "inhomogeneity parameter," and n is numbers of components (36,37). Simulation of the power saturation and redox titration curves was performed using the software Origin (MicroCal Software, Inc.) using the Marquardt-Lovenberg algorithm and simplex method for nonlinear least-square fitting.…”

Section: Methodsmentioning

confidence: 99%

An Escherichia coli Mutant Quinol:Fumarate Reductase Contains an EPR-detectable Semiquinone Stabilized at the Proximal Quinone-binding Site

Hägerhäll¹,

Magnitsky²,

Sled³

et al. 1999

Journal of Biological Chemistry

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The EPR and thermodynamic properties of semiquinone (SQ) species stabilized by mammalian succinate: quinone reductase (SQR) in situ in the mitochondrial membrane and in the isolated enzyme have been well documented. The equivalent semiquinones in bacterial membranes have not yet been characterized, either in SQR or quinol:fumarate reductase (QFR) in situ. In this work, we describe an EPR-detectable QFR semiquinone using Escherichia coli mutant QFR (FrdC E29L) and the wild-type enzyme. The SQ exhibits a g ‫؍‬ 2.005 signal with a peak-to-peak line width of ϳ1.1 milliteslas at 150 K, has a midpoint potential (E m(pH 7.2) ) of ؊56.6 mV, and has a stability constant of ϳ1.2 ؋ 10 ؊2 at pH 7.2. It shows extremely fast spin relaxation behavior with a P 1/2 value of > >500 milliwatts at 150 K, which closely resembles the previously described SQ species (SQ s ) in mitochondrial SQR. This SQ species seems to be present also in wildtype QFR, but its stability constant is much lower, and its signal intensity is near the EPR detection limit around neutral pH. In contrast to mammalian SQR, the membrane anchor of E. coli QFR lacks heme; thus, this prosthetic group can be excluded as a spin relaxation enhancer. The trinuclear iron-sulfur cluster FR3 in the [3Fe-4S] 1؉ state is suggested as the dominant spin relaxation enhancer of the SQ FR spins in this enzyme. E. coli QFR activity and the fast relaxing SQ species observed in the mutant enzyme are sensitive to the inhibitor 2-nheptyl-4-hydroxyquinoline N-oxide (HQNO). In wildtype E. coli QFR, HQNO causes EPR spectral line shape perturbations of the iron-sulfur cluster FR3. Similar spectral line shape changes of FR3 are caused by the FrdC E29L mutation, without addition of HQNO. This indicates that the SQ and the inhibitor-binding sites are located in close proximity to the trinuclear iron-sulfur cluster FR3. The data further suggest that this site corresponds to the proximal quinone-binding site in E. coli QFR.Succinate:quinone reductase (SQR) 1 and quinol:fumarate reductase (QFR) are structurally and functionally similar enzymes with an interesting evolution (1-3). 1ϩ,0 clusters are called S1 or FR1, S2 or FR2, and S3 or FR3 in SQR and QFR, respectively. The membrane anchor domain of the enzyme is more variable and may consist of one or two hydrophobic polypeptides (SdhC/FrdC and SdhD/ FrdD) and contain zero, one, or two b hemes depending on the enzyme species. When two hemes are present, they are denoted heme b H and heme b L . The primary sequence similarity is also much lower in this part of the enzyme. Nevertheless, accumulated evidence indicates that the membrane anchors have a conserved general structure (3, 4). One exception is a group of SQRs lacking the membrane domain and instead containing two different, more or less hydrophilic subunits (5).The membrane-bound enzymes catalyze the oxidation of succinate or the reduction of fumarate in the bacterial cytoplasm or mitochondrial matrix and the reduction or oxidation of quinone/quinol in the membrane. It should be emphasi...

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“…As can be seen in the saturation plot (Fig. 4), the S = 1/2 species is relatively fast relaxing, which is normal for these types of clusters [23]. Therefore it is unlikely that it is a W 5 signal.…”

Section: The [4fe-4s]mentioning

confidence: 80%

Pyrococcus furiosus glyceraldehyde 3‐phosphate oxidoreductase has comparable W^6+/5+ and W^5+/4+ reduction potentials and unusual [4Fe‐4S] EPR properties

Hagedoorn¹,

Freije²,

Hagen³

1999

FEBS Letters

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Pyrococcus furiosus glyceraldehyde 3-phosphate oxidoreductase has been characterized using EPR-monitored redox titrations. Two different W signals were found. W 5 1 is an intermediate species in the catalytic cycle, with the midpoint potentials E m (W 6 a5 ) = 3 3507 mV and E m (W 5 a4 ) = 3 3491 mV. W 5 2 represents an inactivated species with E m (W 6 a5 ) = 3 3329 mV. The cubane cluster exhibits both S = 3/2 and S = 1/2 signals with the same midpoint potential : E m ([4Fe-4S] 2 a1 ) = 3 3335 mV. The S = 1/2 EPR signal is unusual with all g values below 2.0. The titration results combined with catalytic voltammetry data are consistent with electron transfer from glyceraldehyde 3-phosphate first to the tungsten center, then to the cubane cluster and finally to the ferredoxin.z 1999 Federation of European Biochemical Societies.

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Electron spin relaxation of iron-sulphur proteins studied by microwave power saturation

Cited by 194 publications

References 32 publications

Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

An Escherichia coli Mutant Quinol:Fumarate Reductase Contains an EPR-detectable Semiquinone Stabilized at the Proximal Quinone-binding Site

Pyrococcus furiosus glyceraldehyde 3‐phosphate oxidoreductase has comparable W^6+/5+ and W^5+/4+ reduction potentials and unusual [4Fe‐4S] EPR properties

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Electron spin relaxation of iron-sulphur proteins studied by microwave power saturation

Cited by 194 publications

References 32 publications

Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

An Escherichia coli Mutant Quinol:Fumarate Reductase Contains an EPR-detectable Semiquinone Stabilized at the Proximal Quinone-binding Site

Pyrococcus furiosus glyceraldehyde 3‐phosphate oxidoreductase has comparable W6+/5+ and W5+/4+ reduction potentials and unusual [4Fe‐4S] EPR properties

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Pyrococcus furiosus glyceraldehyde 3‐phosphate oxidoreductase has comparable W^6+/5+ and W^5+/4+ reduction potentials and unusual [4Fe‐4S] EPR properties