Temperature jump relaxation kinetics of the P-450cam spin equilibrium

The ferric spin-state equilibrium and relaxation rate of cytochrome P-450 has been examined with temperature jump spectroscopy using a number of camphor analogues known to induce different mixed spin states in the substrate-bound complexes [Gould, P., Gelb, M., & Sligar, S. G. (1981) J. Biol. Chem. 256, 6686]. All temperature-induced spectral changes were monophasic, and the spin-state relaxation rate reached a limiting value at high substrate concentrations. The ferric spin equilibrium constant, Kspin, is defined in terms of the rate constants k1 and k-1 via Kspin = k1/k-1 = [P-450(HS)]/[P-450(LS)] where HS and LS represent high-spin (S = 5/2) and low-spin (S = 1/2) ferric iron, respectively, and the spectrally observed spin-state relaxation rate by kobsd = k1 + k-1. A strong correlation between the fraction of high-spin species and the rate constant, k-1, is observed. For a 3 degrees C temperature jump (from 10 to 13 degrees C), the 23% high-spin tetramethylcyclohexanone complex (Kd = 45 +/- 20 microM) is characterized by a ferric spin relaxation rate of kobsd = 1990 s-1, while the rates for the d-fenchone (41% high spin, Kd = 42 +/- 10 microM) and kobsd = 1990 s-1, while the rates for the d-fenchone (41% high spin, Kd = 42 +/- 10 microM) and camphoroquinone (75% high spin, Kd = 15 +/- 5 microM) complexes are 1430 and 346 s-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulsed EPR Determination of Water Accessibility to Spin-Labeled Amino Acid Residues in LHCIIb

Membrane proteins reside in a structured environment in which some of their residues are accessible to water, some are in contact with alkyl chains of lipid molecules, and some are buried in the protein. Water accessibility of residues may change during folding or function-related structural dynamics. Several techniques based on the combination of pulsed electron paramagnetic resonance (EPR) with site-directed spin labeling can be used to quantify such water accessibility. Accessibility parameters for different residues in major plant light-harvesting complex IIb are determined by electron spin echo envelope modulation spectroscopy in the presence of deuterated water, deuterium contrast in transversal relaxation rates, analysis of longitudinal relaxation rates, and line shape analysis of electron-spin-echo-detected EPR spectra as well as by the conventional techniques of measuring the maximum hyperfine splitting and progressive saturation in continuous-wave EPR. Systematic comparison of these parameters allows for a more detailed characterization of the environment of the spin-labeled residues. These techniques are applicable independently of protein size and require ∼10-20 nmol of singly spin-labeled protein per sample. For a residue close to the N-terminus, in a domain unresolved in the existing x-ray structures of light-harvesting complex IIb, all methods indicate high water accessibility.     

Determination of interspin distances between spin labels attached to insulin: comparison of electron paramagnetic resonance data with the X-ray structure

A method was developed to determine the interspin distances of two or more nitroxide spin labels attached to specific sites in proteins. This method was applied to different conformations of spin-labeled insulins. The electron paramagnetic resonance (EPR) line broadening due to dipolar interaction is determined by fitting simulated EPR powder spectra to experimental data, measured at temperatures below 200 K to freeze the protein motion. The experimental spectra are composed of species with different relative nitroxide orientations and interspin distances because of the flexibility of the spin label side chain and the variety of conformational substates of proteins in frozen solution. Values for the average interspin distance and for the distance distribution width can be determined from the characteristics of the dipolar broadened line shape. The resulting interspin distances determined for crystallized insulins in the R6 and T6 structure agree nicely with structural data obtained by x-ray crystallography and by modeling of the spin-labeled samples. The EPR experiments reveal slight differences between crystal and frozen solution structures of the B-chain amino termini in the R6 and T6 states of hexameric insulins. The study of interspin distances between attached spin labels can be applied to obtain structural information on proteins under conditions where other methods like two-dimensional nuclear magnetic resonance spectroscopy or x-ray crystallography are not applicable.     

Phase memory relaxation times of spin labels in human carbonic anhydrase II: pulsed EPR to determine spin label location.

Phase memory relaxation times (T(M) or T(2)) of spin labels in human carbonic anhydrase II (HCA II) are reported. Spin labels (N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide, IPSL) were introduced at cysteines, by site-directed mutagenesis at seven different positions in the protein. By two pulse electron paramagnetic resonance (EPR), electron spin echo decays at 45 K are measured and fitted by stretched exponentials, resulting in relaxation parameters T(M) and x. T(M) values of seven positions are between 1.6 micros for the most buried residue (L79C) and 4.7 micros for a residue at the protein surface (W245C). In deuteriated buffer, longer T(M) are found for all but the most buried residues (L79C and W97C), and electron spin echo envelop modulation (ESEEM) of deuterium nuclei is observed. Different deuterium ESEEM patterns for W95C and W16C (surface residue) indicate differences in the local water concentration, or accessibility, of the spin label by deuterium. We propose T(M) as a parameter to determine the spin label location in proteins. Furthermore, these systems are interesting for studying the pertaining relaxation mechanism.     

Direct spectroscopic evidence for the presence of a 6Fe cluster in an iron-sulfur protein isolated from Desulfovibrio desulfuricans (ATCC 27774)

A novel iron-sulfur protein was purified from the extract of Desulfovibrio desulfuricans (ATCC 27774) to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a monomer of 57 kDa molecular mass. It contains comparable amounts of iron and inorganic labile sulfur atoms and exhibits an optical spectrum typical of iron-sulfur proteins with maxima at 400, 305, and 280 nm. M?ssbauer data of the as-isolated protein show two spectral components, a paramagnetic and a diamagnetic, of equal intensity. Detailed analysis of the paramagnetic component reveals six distinct antiferromagnetically coupled iron sites, providing direct spectroscopic evidence for the presence of a 6Fe cluster in this newly purified protein. One of the iron sites exhibits parameters (delta EQ = 2.67 +/- 0.03 mm/s and delta = 1.09 +/- 0.02 mm/s at 140 K) typical for high spin ferrous ion; the observed large isomer shift indicates an iron environment that is distinct from the tetrahedral sulfur coordination commonly observed for the iron atoms in iron-sulfur clusters and is consistent with a penta- or hexacoordination containing N and/or O ligands. The other five iron sites are most probably high spin ferric. Three of them show parameters characteristic for tetrahedral sulfur coordination. In correlation with the EPR spectrum of the as-purified protein which shows a resonance signal at g = 15.3 and a group of signals between g = 9.8 and 5.4, this 6Fe cluster is assigned to an unusual spin state of 9/2 with zero field splitting parameters D = -1.3 cm-1 and E/D = 0.062. Other EPR signals attributable to minor impurities are also observed at the g = 4.3 and 2.0 regions. The diamagnetic M?ssbauer component represents a second iron cluster, which, upon reduction with dithionite, displays an intense S = 1/2 EPR signal with g values at 2.00, 1.83, and 1.31. In addition, an EPR signal of the S = 3/2 type is also observed for the dithionite-reduced protein.     

Determining Rieske cluster reduction potentials

The Rieske iron-sulfur proteins have reduction potentials ranging from -150 to +400 mV. This enormous range of potentials was first proposed to be due to differing solvent exposure or even protein structure. However, the increasing number of available crystal structures for Rieske iron-sulfur proteins has shown this not to be the case. Colbert and colleagues proposed in 2000 that differences in the electrostatic environment, and not structural differences, of a Rieske proteins are responsible for the wide range of reduction potentials observed. Using computational simulation methods and the newly determined structure of Pseudomonas sp. NCIB 9816-4 naphthalene dioxygenase Rieske ferredoxin (NDO-F9816-4), we have developed a model to predict the reduction potential of Rieske proteins given only their crystal structure. The reduction potential of NDO-F9816-4, determined using a highly oriented pyrolytic graphite electrode, was -150+/-2 mV versus the standard hydrogen electrode. The predicted reduction potentials correlate well with experimentally determined potentials. Given this model, the effect of protein mutations can be evaluated. Our results suggest that the reduction potential of new proteins can be estimated with good confidence from 3D structures of proteins. The structure of NDO-F9816-4 is the most basic Rieske ferredoxin structure determined to date. Thus, the contributions of additional structural motifs and their effects on reduction potential can be compared with respect to this base structure.     

A new plant-type ferredoxin from halobacteria.

A stable, 2Fe-type ferredoxin has been prepared from Halobacterium halobium and purified by chromatography. A similar ferredoxin was also found in three other Halobacteria. The ferredoxin is present in large amounts-about 1 percent of the total soluble protein. From amino acid composition a molecular weight of 14800 +/- 200 was calculated. The ferredoxin was found to contain two atoms each of iron and sulphide. The midpoint redox potential of the protein is about -345 mV. The electron paramagnetic resonance spectrum of the reduced form shows much similarity to plant and algal ferredoxins with gx = 1.90, gy = 1.97 and gz = 2.07. The same similarity is observed in the optical absorption, optical rotatory dispersion and circular dichroism spectra. However it does not seem to mediate electron transport in the NADP-photoreduction system of chloroplasts. Extracts of the bacterial cells catalyze the reduction of the ferredoxin by NADH.     

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.     

High-multiplicity spin states of 2[4Fe-4Se]+ clostridial ferredoxins

The electron paramagnetic resonance (EPR) spectra of the reduced selenium-substituted 2-[4Fe-4Se]+ ferredoxins from three bacteria of the Clostridium genus display low-field signals at g = 5.17, g = 10.11, and g = 12.76. The positions, shapes, and temperature dependencies of these signals have allowed their assignments to the three excited states of an S = 7/2 spin multiplet, the fundamental state of which is observed as unusual features in low-temperature (T less than or equal to 20 K) M?ssbauer spectra. The S = 7/2 spin state is present in 2[4Fe-4Se]+ clostridial ferredoxins together with the classical S = 1/2 state and with a S = 3/2 state, the fundamental doublet of which is observed as a broad signal in the g = 3-4 region. The relative intensities of the EPR signals corresponding to these spin states depend on the species of Clostridium that the ferredoxin is extracted from. In contrast with clostridial ferredoxins, the reduced selenium-substituted ferredoxin from Bacillus stearothermophilus, which differs significantly from the clostridial proteins by its primary structure and by its containing only one tetranuclear cluster, displays only the S = 1/2 state. Thus, the high-multiplicity spin states arise from a specific interaction between the clostridial ferredoxin polypeptide chain and the reduced [4Fe-4Se]+ clusters.     

Purification and properties of ferredoxin and rubredoxin from Butyribacterium methylotrophicum.

A ferredoxin and a rubredoxin from Butyribacterium methylotrophicum, which displays a carbonyl-dependent acetyl-coenzyme A synthesis, were purified to electrophoretic homogeneity. The two electron carriers showed absorption spectra similar to those in Clostridium species. The ferredoxin displayed absorption peaks at 280 and 391 nm, while rubredoxin displayed absorption peaks at 279, 382, and 482 nm. Minimum molecular weights calculated from the respective amino acid compositions were 5,727 for ferredoxin and 5,488 for rubredoxin, excluding iron and inorganic sulfur atoms. Both electron carriers were isolated as monomers, according to gel-filtration data. Electron spin resonance analysis revealed that the ferredoxin was a 2[4Fe-4S]-type and that both clusters had a midpoint redox potential value of -410 mV, whereas rubredoxin contained one acid-stable iron and had a redox value of -40 mV. The coupling of these electron carriers to hydrogenase and carbon monoxide dehydrogenase activities was investigated. Rubredoxin showed higher activity towards carbon monoxide dehydrogenase, whereas ferredoxin showed higher activity towards hydrogenase.     

The three-iron cluster in a ferredoxin from Desulphovibrio gigas. A low-temperature magnetic circular dichroism study

Ferredoxin II from Desulphovibrio gigas is a tetrameric protein containing a novel iron-sulphur cluster consisting of three iron atoms. The low-temperature magnetic circular dichroism (MCD) spectra of the oxidized and dithionite-reduced forms of ferredoxin II have been measured over the wavelength range approx. 300-800 nm. Both oxidation levels of the cluster are shown to be paramagnetic, although only the oxidized form gives an EPR signal. MCD magnetization curves have been constructed over the temperature range approx. 1.5-150 K and at fields between 0 and 5.1 Tesla. The curve for the oxidized protein can be fitted to a ground state of spin S = 1/2 with an isotropic g factor of 2.01. There is evidence for the thermal population of a low-lying electronic state above 50 K. The reduced protein gives a distinctive set of magnetization curves that are tentatively assigned to a ground state of S = 2, with a predominantly axial zero-field distortion that leaves the doublet Ms = +/-2 lowest in energy. The zero-field components have a maximum energy spread of approx. 15 cm-1. which places an upper limit of 4 cm-1 on the axial zero-field parameter D. The MCD spectra of the oxidized and reduced forms of the cluster are quite distinctive from one another. The spectra of the oxidized state are also different from those of oxidized high-potential iron protein from Chromatium and should provide a useful criterion for distinguishing between four- and three-iron clusters in their highest oxidation levels.     

Evidence for the presence of a [2Fe-2S] ferredoxin in bean sprouts

An iron-sulfur protein with properties similar to those of ferredoxins found in the leaves of higher plants has been isolated from bean sprouts--a non-photosynthetic plant tissue. The bean sprout protein has a molecular mass of 12.5 kDa and appears to contain a single [2Fe-2S] cluster. The absorbance and circular dichroism spectra of the bean sprout protein resemble those of spinach leaf ferredoxin and the bean sprout protein can replace spinach ferredoxin as an electron donor for NADP+ reduction, nitrite reduction and thioredoxin reduction by spinach leaf enzymes. Although the reduced bean sprout protein (Em = -440 mV) is a slightly stronger reductant than spinach ferredoxin and appears to be less acidic than spinach ferredoxin, the two proteins are similar enough so that the bean sprout protein is recognized by an antibody raised against spinach ferredoxin.     

Electron spin relaxation time of Fe(III) in high spin heme proteins.

The electron spin relaxation time of high spin Fe(III), taus, was determined from the frequency dependence (5-100 MHz) of the longitudinal proton relaxation rates of water in solutions of catalase, metmyoglobin and acid ferricytochrome c. In all three high-spin heme proteins the relaxation rates incrased below 25 MHz, while no frequency dependence was observed above that frequency. The results are interpreted by assuming that taus, which modulates the dipolar interaction between the unpaired electrons of the iron and the water protons, is frequently independent. Its value was determined to be (6 +/- 1) - 10(-11) s.     

Purification and characterization of a ferredoxin from Rhizobium japonicum bacteroids

An eight-iron, eight-sulfur ferredoxin from Rhizobium japonicum bacteroids of soybean root nodules has been purified to apparent homogeneity as judged by disc gel electrophoresis. The purification procedure included chromatography on DEAE-cellulose, Bio-Gel P-60, and hydroxylapatite. Specific activities of several purified preparations of bacteroid ferredoxin ranged from 1700 to 1900 nmol of C2H4 produced . min-1 . mg-1 in the reaction mediating electron transfer between illuminated chloroplasts and bacteroid nitrogenase. A molecular weight of 6740 for the protein was determined by low speed sedimentation equilibrium and a molecular weight of 6500 was estimated from the mobility of bacteroid ferredoxin relative to the mobility of standard proteins during sodium dodecyl sulfate disc gel electrophoresis. All of the common amino acids were present except arginine, methionine, and tryptophan. The absorbance spectrum of the oxidized protein exhibited maxima at 285 nm and 380 nm with a shoulder near 305 nm. The A380/A285 ratio was 0.76 and the extinction coefficient at 380 nm for the oxidized protein was found to be 30,800 M-1. Equilibration of bacteroid ferredoxin with methyl viologen at various potentials revealed a midpoint oxidation-reduction potential of -484 mV. Spectrophotometric examination of iron-sulfur clusters extruded from bacteroid ferredoxin with benzenethiol and the transfer of its iron-sulfur clusters to other ferredoxins established the presence of two [4Fe-4S] clusters in a molecule of bacteroid ferredoxin. The EPR spectrum of oxidized ferredoxin consisted of a small signal at g = 2.02 integrating to 0.19 spin/molecule. The EPR spectrum of ferredoxin reduced with 5-deazaflavin exhibited a signal with features at g values of 1.88, 1.94, 2.01, and 2.07, and integrated to 1.7 spins/molecule. The EPR properties of bacteroid ferredoxin are characteristic of a ferredoxin operating between the 1+ and 2+ oxidation levels. Bacteroid ferredoxin mediated electron transfer to clostridial hydrogenase, but was not reduced by the clostridial phosphoroclastic system in the presence of pyruvate. Bacteroid ferredoxin reduced by illuminated 5-deazariboflavin also supported a high rate of C2H2 reduction by bacteroid nitrogenase which was free of Na2S2O4. It was concluded, on this basis, that bacteroid ferredoxin has the capability of functioning as the electron donor for nitrogenase in R. japonicum.     

Protein control of iron-sulfur cluster redox potentials.

The relationship between the three-dimensional structures of iron-sulfur proteins and the redox potentials of their iron-sulfur clusters is of fundamental importance. We report calculations of the redox potentials of the [Fe4S4(S-cys)4]-2/-3 couple in four crystallographically characterized proteins: Azotobacter vinelandii ferredoxin I, Peptococcus aerogenes ferredoxin, Bacillus thermoproteolyticus ferredoxin, and Chromatium vinosum high potential iron protein (HiPIP). Our calculations use the "protein dipoles Langevin dipoles" microscopic electrostatic model, which includes both protein and solvent water. The variations in calculated redox potentials are in excellent agreement with experimental data. In particular, our results confirm the important role of amide groups close to the cluster in separating the potential of C. vinosum HiPIP from those of the other three proteins. However, the potentials of these latter exhibit a substantial range despite extremely similar amide group environments of their clusters. Our results show that the potentials in these proteins are tuned in part by varying the access of solvent water to the neighborhood of the cluster. Our calculations provide the first successful quantitative modeling of the protein control of iron-sulfur cluster redox potentials.     

The electron spin relaxation of the electron acceptors of Photosystem I reaction centre studied by microwave power saturation

Photosystem I particles from spinach were reduced by illumination at 77 K. Under these conditions the one-electron transfer from P-700 resulted in a reduction of only one acceptor molecule of the reaction centre. The EPR signals at g = 2.05, 1.94 and 1.86 were attributed to reduced centre A and the smaller signals at g = 2.07, 1.92 and 1.89 to reduced centre B. Reduction of both centres by dithionite in the dark lead to signals at g = 2.05, 1.99, 1.96, 1.94, 1.92 and 1.89. Thus, the features at g = 2.07 and 1.86 disappeared and new signals at g = 1.99 and 1.96 were observed. From the spectral changes it followed that the iron-sulphur centres A and B interact magnetically. Temperature dependent EPR spectra demonstrated a faster electron spin relaxation of centre A than of centre B.

These conclusions were corroborated using microwave power saturation of the respective EPR signals. The saturation data of the fully reduced centres A and B could not be fitted using the saturation equation for a one-electron spin system. The magnetic interaction between the [4Fe-4S] centres of the electron acceptors A and B resulted in saturation properties which are similar to those of the 2[4Fe-4S] ferredoxin from Clostridium pasteurianum.

For centre X a high proportion of homogeneous broadening of the EPR lines was inferred from the inhomogeneity parameter (b = 1.83). It was, therefore, concluded that centre X is most probably an anion radical of chlorophyll. From the low temperature necessary for observing the EPR signal of centre X followed that the drastic relaxation enhancement has to be attributed to a magnetic interaction of the anion radical with iron.     

The electron spin relaxation of the electron acceptors of photosystem I reaction centre studied by microwave power saturation.

Photosystem I particles from spinach were reduced by illumination at 77 K. Under these conditions the one-electrom transfer from P-700 resulted in a reduction of only one acceptor molecule of the reaction centre. The EPR signals at g=2.05, 1.94 and 1.86 were attributed to reduced centre A and the smaller signals at g=2.07, 1.92 and 1.89 to reduced centre B. Reduction of both centres by dithionite in the dark lead to signals at g=2.05, 1.99, 1.96, 1.94, 1.92 and 1.89. Thus, the features at g=2.07 and 1.86 disappeared and new signals at g=1.99 and 1.96 were observed. From the spectral changes it followed that the iron-sulphur centres A and B interact magnetically. Temperature dependent EPR spectra demonstrated a faster electron spin relaxation of centre A than of centre B. These conclusions were corroborated using microwave power saturation of the respective EPR signals. The saturation data of the fully reduced centres A and B could not be fitted using the saturation equation for a one-electron spin system. The magnetic interaction between the (4Fe-4S) CENTRes of the electron acceptors A and B resulted in saturation properties which are simular to those of the 2(4Fe-4S) ferredoxin from Clostridium pasteurianum. For centre X a high proportion of homogeneous broadening of the EPR lines was inferred from the inhomogeneity parameter (b=1.83). It was, therefore, concluded that centre X is most probably an anion radical of chlorophyll. From the low temperature necessary for observing the EPR signal of centre X followed that the drastic relaxation enhancement has to be attributed to a magnetic interaction of the anion radical with iron.     

The coordination of imidazole and substituted pyridines by the hemeoctapeptide N-acetyl-ferromicroperoxidase-8 (FeIINAcMP8)

The N-terminus acetylated ferric hemeoctapeptide from cytochrome c, N-acetylmicroperoxidase-8 (Fe(III)-NAcMP8) can be reduced by dithionite in aqueous solution to produce Fe(II)-NAcMP8. The UV-Vis spectrum has a broad Soret band and relatively poorly defined Q bands which is consistent with a mixture of a five-coordinate high spin species with His as the axial ligand and a six-coordinate, predominantly high spin species with His/H(2)O as axial ligands. There are two spectroscopically observable pK(a)s at 8.7+/-0.1 and 10.9+/-0.2 which are attributed to ionization of a heme propionic acid group and coordinated H(2)O, respectively; a pK(a) > or = 14 is due to ionization of the proximal His ligand. Equilibrium constants were determined by UV-Vis spectrophotometry at 25.0+/-0.2 degrees C and 0.5 M ionic strength (NaClO(4)) for the coordination of imidazole and a number of substituted pyridines, and complement available data for the ferric hemepeptide, allowing a comparison to be made of the affinity of an iron porphyrin with Fe in the +2 and +3 oxidation states towards these ligands. Imidazole is coordinated more strongly by the ferric porphyrin (log K=4.08) than by the ferrous porphyrin (log K=3.40). The equilibrium constants for coordination of pyridines by the ferric and ferrous porphyrins increase and decrease, respectively, with increasing ligand basicity. Values determined by cyclic voltammetry show the same dependence on the identity of the ligand. In the ferric porphyrin, the stability of the complex increases with the basicity of the ligand and hence its ability to donate electron density onto the metal. In the case of the more electron rich ferrous porphyrin, greater stability occurs with pyridine ligands that have an electron withdrawing group and hence can accept electron density from the metal. This is consistent with the midpoint reduction potentials E(1/2) of the pyridine complexes determined by cyclic voltammetry; E(1/2) is linearly dependent on, and becomes more negative with an increase in, ligand basicity. Log K for coordination of pyridines by the ferrous hemepeptide correlates well with the energy of the ligand frontier orbital with pi symmetry, suggesting that pi-bonding effects are significant in determining the strength of binding of pyridines by a ferrous porphyrin.     

Purification and spectropotentiometric characterization of Escherichia coli NrfB, a decaheme homodimer that transfers electrons to the decaheme periplasmic nitrite reductase complex

Escherichia coli can reduce nitrite to ammonium via a 120-kDa decaheme homodimeric periplasmic nitrite reductase (NrfA) complex. Recent structure-based spectropotentiometric studies are shedding light on the catalytic mechanism of NrfA; however, electron input into the enzyme has not been addressed biochemically. This study reports the first purification of NrfB, a novel 20-kDa pentaheme c-type cytochrome encoded by the nrfB gene that follows the nrfA gene in many bacterial nrf operons. Analyses by gel filtration demonstrated that NrfB purifies as a decaheme homodimer. Analysis of NrfB by UV-visible and magnetic circular dichroism spectroscopy demonstrates that all five NrfB ferric heme irons are low spin and are most likely coordinated by two axial histidine ligands. Spectropotentiometry revealed that the midpoint redox potentials of five ferric hemes were in the low potential range of 0 to -400 mV. Analysis by low temperature EPR spectroscopy revealed signals that arise from two classes of bis-His ligated low spin hemes, namely a rhombic trio at g(1,2,3) = 2.99, 2.27, and 1.5 that arises from two hemes in which the planes of histidine imidazole rings are near-parallel and a large g(max) signal at g = 3.57 that arises from three hemes in which the planes of the histidine imidazole rings are near-perpendicular. NrfB was also overexpressed as a recombinant protein, which had similar spectropotentiometric properties as the native protein. Reconstitution experiments demonstrated that the reduced decaheme NrfB dimer could serve as a direct electron donor to the oxidized decaheme NrfA dimer, thus forming a transient 20-heme [NrfB](2)[NrfA](2) electron transfer complex.     

Electron spin relaxation of CuA and cytochrome a in cytochrome c oxidase. Comparison to heme, copper, and sulfur radical complexes

The method of continuous saturation has been used to measure the electron spin relaxation parameter T1T2 at temperatures between 10 and 50 K for a variety of S = 1/2 species including: CuA and cytochrome a of cytochrome c oxidase, the type 1 copper in several blue copper proteins, the type 2 copper in laccase, inorganic Cu(II) complexes, sulfur radicals, and low spin heme proteins. The temperature dependence and the magnitude of T1T2 for all of the species examined are accounted for by assuming that the Van Vleck Raman process dominates the electron spin-lattice relaxation. Over the entire temperature range examined, the relaxation of the type 1 coppers in six to seven times faster than that of type 2 copper, inorganic copper, and sulfur radicals, in spite of the similar g-anisotropies of these species. This result may indicate that the coupling of the phonon bath to the spin center is more effective in type 1 coppers than in the other complexes studied. The relaxation of CuA of cytochrome oxidase exhibits an unusual temperature dependence relative to the other copper complexes studied, suggesting that the protein environment of this center is different from that of the other copper centers studied and/or that CuA is influenced by a magnetic dipolar interaction with another, faster-relaxing paramagnetic site in the enzyme. A comparison of the saturation characteristics of the CuA EPR signal in native and partially reduced CO complexes of the enzyme also suggests the existence of such an interaction. The implications of these results with respect to the disposition of the metal centers in cytochrome oxidase are discussed.