Elucidation of a [4Fe-4S] cluster degradation pathway: rapid kinetic studies of the degradation of Chromatium vinosum HiPIP.

Irreversible disassembly of the 4Fe-4S cluster in Chromatium vinosum high-potential iron protein (HiPIP) has been investigated in the presence of a low concentration of guanidinium hydrochloride. From the dependence of degradation rate on [H+], it is deduced that at least three protons are required to trigger efficient cluster degradation. Under these conditions the protonated cluster shows broadened Mössbauer signals, but delta EQ (1.1 mm/s) and delta (0.44 mm/s) are similar to the native form. Collapse of the protonated transition state complex, revealed by rapid-quench Mössbauer experiments, occurs with a measured rate constant kobs approximately 0.72 +/- 0.35 s-1 that is consistent with results from time-resolved electronic absorption and fluorescence (kobs approximately 0.4 +/- 0.1 s-1) and EPR (kobs approximately 0.62 +/- 0.18 s-1) measurements. Apparently, guanidinium hydrochloride serves to perturb the tertiary structure of the protein, facilitating protonation of the cluster, but not degradation per se. Release of iron ions occurs even more slowly with kobs approximately 0.07 +/- 0.02 s-1, as determined by the appearance of the g = 4.3 EPR signal. Proton-mediated cluster degradation is sensitive to the oxidation state of the cluster, with the oxidized state showing a two-fold slower rate in acidic solutions as a result of increased electrostatic repulsion with the cluster. Consistent results are obtained from absorption, fluorescence, Mössbauer and EPR measurements.

Electrochemical and spectroscopic properties of the iron-sulfur flavoprotein from Methanosarcina thermophila.

An iron-sulfur flavoprotein (Isf) from the methanoarchaeaon Methanosarcina thermophila, which participates in electron transfer reactions required for the fermentation of acetate to methane, was characterized by electrochemistry and EPR and Mössbauer spectroscopy. The midpoint potential (Em) of the FMN/FMNH2 couple was -0.277 V. No flavin semiquinone was observed during potentiometric titrations; however, low amounts of the radical were observed when Isf was quickly frozen after reaction with CO and the CO dehydrogenase/acetyl-CoA synthase complex from M. thermophila. Isf contained a [4Fe-4S]2+/1+ cluster with g values of 2.06 and 1.93 and an unusual split signal with g values at 1.86 and 1.82. The unusual morphology was attributed to microheterogeneity among Isf molecules. The Em value for the 2+/1+ redox couple of the cluster was -0.394 V. Extracts from H2-CO2-grown Methanobacterium thermoautotrophicum cells catalyzed either the H2- or CO-dependent reduction of M. thermophila Isf. In addition, Isf homologs were found in the genomic sequences of the CO2-reducing methanoarchaea M. thermoautotrophicum and Methanococcus jannaschii. These results support a general role for Isf in electron transfer reactions of both acetate-fermenting and CO2-reducing methanoarchaea. It is suggested that Isf functions to couple electron transfer from ferredoxin to membrane-bound electron carriers, such as methanophenazine and/or b-type cytochromes.

Cyanide binding and active site structure in heme-copper oxidases: normal coordinate analysis of iron-cyanide vibrations of a3(2+)CN- complexes of cytochromes ba3 and aa3.

The cyanide isotope-sensitive low-frequency vibrations of ferrous cyano complexes of cytochrome a3 are studied for cytochrome ba3 from Thermus thermophilus and cytochrome aa3 from bovine heart. Cyanide complexes of ba3 display three isotope sensitive frequencies at 512, 485, and 473 cm-1. The first is primarily an Fe-C stretching motion, whereas the lower wavenumber modes are bending motions. These iron-cyanide vibrations are independent of the redox levels of the other metal centers in the protein. On the other hand, the fully reduced bovine derivative complexed with cyanide gives rise to a bending vibration at 503 cm-1 and a stretching vibration at 469 cm-1. That is, the ordering of the stretching and bending frequencies is reversed from that of the bacterial protein. These results are analyzed by normal coordinate calculations to obtain comparative models for the binuclear O2 reducing site of the two proteins. We find that the observed frequencies are consistent with a linear Fe-C-N group and larger Fe-C stretching force constant (2.558 mdyn/A) for ba3 and a slightly bent Fe-C-N group (angle approximately 170 degrees) and a smaller Fe-C stretching force constant (2.335 mdyn/A) for aa3. Thus, there are significant differences in the interaction of cyanide with ferrous a3 in the two proteins that are most likely caused by a weaker proximal histidine interaction and stronger peripheral heme electron withdrawing effects in ba3. Possible sources of these protein-induced effects are discussed. Using the analysis developed here, comparison of the FeCN stretching and bending frequencies of the ferrous bovine a3-CN complex to those obtained from the ferric a3-CN complex suggests that upon conversion of the resting to the fully reduced protein, a conformational change occurs that constrains the ligand binding site.

Further characterization of the spin coupling observed in oxidized hydrogenase from Chromatium vinosum. A Mössbauer and multifrequency EPR study.

Hydrogenase from Chromatium vinosum contains 1 Ni, 11-12 Fe, and ca. 9 sulfides. EPR and Mössbauer studies of the enzyme prepared in four different oxidation states show that the enzyme contains two Fe4S4 and one Fe3S4 cluster. In the oxidized (2+) state, the Mössbauer parameters of the two Fe4S4 clusters are typical for this cluster type. Upon reduction, however, these clusters do not exhibit the familiar g = 1.94 signal. The unusual nature of the reduced clusters is also borne out by the Mössbauer spectra which exhibit fairly small magnetic hyperfine interactions similar to those of centers I and II of the Desulfovibrio gigas enzyme. The Mössbauer spectra of the Fe3S4 cluster in the oxidized (1+) and reduced states are typical for this cluster type. The C. vinosum hydrogenase undergoes a reversible redox reaction at Em = +150 mV (vs NHE). Above +150 mV the EPR spectra exhibit signals (previously called signals 2 and 4) that reflect a weak interaction between Ni(III) and an Fe-containing moiety. By clamping the Ni in the diamagnetic Ni(II).CO form, we have discovered that signal 2 (X-band resonances at g = 2.01, 1.974, and 1.963) involves the Fe3S4 cluster and an as yet unidentified paramagnetic moiety. The "coupled" system exhibits magnetic hyperfine interactions quite different from those of the uncoupled [Fe3S4]1+ cluster. We have not yet been able to assign a spin to the coupled state but some of the features of the state are reminiscent of an S = 1 system. The Mössbauer data suggest, but do not prove, that an extra Fe site may be present that shuttles between low-spin Fe(III) and low-spin Fe(II) with Em = +150 mV. The Fe(III) may be located between the Ni(III) and the Fe3S4 cluster enabling it to mediate the interaction between the cluster and the Ni site. In this picture, the Fe(III) site is part of the coupled state that gives rise to signal 2. Other possibilities for signal 2 involve a ligand-based oxidation of the [Fe3S4]1+ cluster or generation of a nearby radical.

Thiol/disulfide formation associated with the redox activity of the [Fe3S4] cluster of Desulfovibrio gigas ferredoxin II. 1H NMR and Mössbauer spectroscopic study.

Desulfovibrio gigas ferredoxin II (FdII) is a small protein (alpha 4 subunit structure as isolated; M(r) approximately 6400 per subunit; 6 cysteine residues) containing one Fe3S4 cluster per alpha-subunit. The x-ray structure of FdII has revealed a disulfide bridge formed by Cys-18 and Cys-42 approximately 13 A away from the center of the cluster; moreover, the x-ray structure indicates that Cys-11 forms a disulfide bridge with a methanethiol. In the oxidized state, FdIIoxm the 1H NMR spectra, exhibit four low-field contact-shifted resonances at 29, 24, 18, and 15.5 ppm whereas the reduced state, FdIIR (S = 2), yields two features at +18.5 and -11 ppm. In the course of studying the redox behavior of FdII, we have discovered a stable intermediate, FdIIint, that yields 1H resonances at 24, 21.5, 21, and 14 ppm. This intermediate appears in the potential range where the cluster (E'0 approximately -130 mV) is reduced from the [Fe3S4]1+ to the [Fe3S4]0 state. FdIIint is observed during reductive titrations with dithionite or hydrogen/hydrogenase or after partial oxidation of FdIIR by 2,6-dichlorophenolindophenol or air. Our studies show that a total of three electrons per alpha-subunit are transferred to FdII. Our experiments demonstrate the absence of a methanethiol-Cys-11 linkage in our preparations, and we propose that two of the three electrons are used for the reduction of the disulfide bridge. Mössbauer (and EPR) studies show that the Fe3S4 cluster of FdIIint is at the same oxidation level as FdIIox, but indicate some changes in the exchange couplings among the three ferric sites. Our data suggest that the differences in the NMR and Mössbauer spectra of FdIIox and FdIIint result from conformational changes attending the breaking or formation of the disulfide bridge. The present study suggests that experiments be undertaken to explore an in vivo redox function for the disulfide bridge.

Spectroscopic characterization of cytochrome ba3, a terminal oxidase from Thermus thermophilus: comparison of the a3/CuB site to that of bovine cytochrome aa3.

Unliganded and cyano derivatives of cytochrome ba3 from Thermus thermophilus have been examined by UV-vis, EPR, and resonance Raman spectroscopies. Species of cytochrome ba3 investigated include its resting, as-isolated, fully oxidized state, the fully reduced, unliganded enzyme, the one-electron-reduced cyano complex, the three-electron-reduced cyano complex, and the fully reduced cyano complex. Results are compared to those obtained from similar adducts of bovine cytochrome aa3, in particular, the fully reduced cyano complex. Our objective was to identify structural similarities and differences at the ligand-binding binuclear site of the two enzymes. We observed that the inner core skeletal vibrations of cytochrome a3 are the same for similar adducts of the bacterial ba3 and mammalian aa3, indicating similar spin and iron-porphyrin coordination properties resulting in comparable porphyrin core geometries. On the other hand, many of the vibrational frequencies associated with the formyl and vinyl peripheral substituents, and the outer pyrrole carbon atoms differ between the bovine and bacterial enzymes. Use of 57Fe labeled ba3 allows identification of two separate vFe-N(His) frequencies displayed by the fully reduced, unliganded cytochrome. These frequencies, occurring at 193 and 209 cm-1, are ascribed to distinct protein conformers, which are best evidenced by the Fe-N(His) vibrations. This result is again in contrast to the bovine enzyme which has been shown by others to display a single Fe-N(His) stretching frequency at 214 cm-1. The low-frequency Fea3(2+)-CN- vibrations of the three-electron and fully reduced cyano complexes of cytochrome ba3 are identified by using 15N and 13C isotopomers of CN-. These spectral signatures are identical to those reported earlier for the one-electron-reduced cyanide adduct (cytochrome a3 reduced), showing that the Fea3(2+)-CN- vibrational frequencies are independent of the redox states of the other three metal centers. Similarly, the CuB2+ EPR signatures appear similar in both the one-electron- and three-electron-reduced cyanide adducts. On the other hand, the electronic absorption spectra of ferrous alpha 3-CN- show systematic red-shifts of the alpha band as each of the other metal centers is reduced, and other, more subtle, differences in the electronic absorptions of the three-electron-reduced and four-electron-reduced cyanide adducts are revealed in the difference spectra. The relevance of these findings toward explaining the different cyanide binding and redox chemistry described herein and toward establishing the extent of structural analogy between the oxygen binding sites of the two proteins is discussed.

Cytochrome caa3 from the thermophilic bacterium Thermus thermophilus: a member of the heme-copper oxidase superfamily.

The subject of this short review is the cytochrome c oxidase (caa3) from the thermophilic bacterium Thermus thermophilus. First, some of the extensive physical and enzymological results obtained with this enzyme are reviewed, and two experiments are described, involving isotope substitutions in combination with Mössbauer and ENDOR spectroscopies, which have provided novel insight into the active sites of the enzyme. Second, we summarize recent molecular genetic work showing that Thermus cytochrome caa3 is a bona fide member of the superfamily of heme-copper oxidases. Finally, we present a rough three-dimensional model and speculate about certain features of the metal-binding sites.

Magnetic circular dichroism study of cytochrome ba3 from Thermus thermophilus: spectral contributions from cytochromes b and a3 and nanosecond spectroscopy of CO photodissociation intermediates.

Near-UV-vis magnetic and natural circular dichroism (MCD and CD) spectra of oxidized, reduced, and carbonmonoxy-complexed cytochrome ba3, a terminal oxidase from the bacterium Thermus thermophilus, and nanosecond time-resolved MCD (TRMCD) and CD (TRCD) spectra of the unligated species formed after photodissociation of the CO complex are presented. The spectral contributions of individual cytochromes b and a3 to the Soret region MCD are identified. TRMCD spectroscopy is used to follow the spin state change (S = 0 to S = 2) in cytochrome a3(2+) following photodissociation of the CO complex. There is prompt appearance of the high-spin state after photolysis, as found previously in mammalian cytochrome oxidase [Goldbeck, R. A., Dawes, T. D., Einarsdóttir, O., Woodruff, W. H., & Kliger, D. S. (1991) Biophys. J. 60, 125-134]. Peak shifts of 1-10 nm appear in the TRMCD, TRCD, and time-resolved UV-vis absorption spectra of the photolyzed enzyme throughout its observable lifetime, indicating that the photolyzed enzyme does not relax to its equilibrium deliganded form before recombination with CO occurs hundreds of milliseconds later. Direct heme-heme interaction is not found in cytochrome ba3, but red-shifts in the MCD and absorption spectra of both cytochromes b and (photolyzed) a3 are correlated with a CO-liganded form of the protein. The long time (tau approximately greater than 1 s) needed for relaxation of the cytochrome b and a3 peaks to their static positions suggests that CO binding to a3 induces a global conformational change in the protein that weakly perturbs the MCD and absorption spectra of b and photolyzed a3. Fea3 binds CO more weakly in cytochrome ba3 than in cytochrome aa3. The MCD spectrum of reduced enzyme solution placed under 1 atm of CO contains a peak at 446 nm that shows approximately 30% of total cytochrome a3 remains pentacoordinate, high-spin.

Reaction of cyanide with cytochrome ba3 from Thermus thermophilus: spectroscopic characterization of the Fe(II)a3-CN.Cu(II)B-CN complex suggests four 14N atoms are coordinated to CuB.

Cytochrome ba3 from Thermus thermophilus reacts slowly with excess HCN at pH 7.4 to create a form of the enzyme in which CuA, cytochrome b, and CuB remain oxidized, while cytochrome a3 is reduced by one electron, presumably with the formation of cyanogen. We have examined this form of the enzyme by UV-visible, resonance Raman, EPR, and electron nuclear double resonance spectroscopies in conjunction with permutations of 13C- and 15N-labeled cyanide. The results support a model in which one CN- binds through the carbon atom to ferrous a3, supporting a low-spin (S = 0) configuration on the Fe; bridging by this cyanide to the CuB is weak or absent. Four 14N atoms, presumably donated by histidine residues of the protein, provide a strong equatorial ligand field about CuB; a second CN- is coordinated through the carbon atom to CuB in an axial position.

Mössbauer study of the inactive Fe3S4 and Fe3Se4 and the active Fe4Se4 forms of beef heart aconitase.

Active beef heart aconitase contains an iron-sulfur cluster with an [Fe4S4]2+ core. This cluster can be converted into Fe3S4 with concomitant loss of enzymatic activity. We have reconstituted apo-aconitase with iron and selenide to obtain Fe4Se4 aconitase. The Se analog has higher catalytic activity than the native S-containing enzyme when isocitrate is the substrate. Oxidation of [Fe4Se4]2+ with ferricyanide yields the inactive [Fe3Se4]1+ form. The Se-containing 3-Fe cluster can be reduced to [Fe3Se4]0. We have studied the [Fe3S4]1+,0, [Fe3Se4]1+,0, and [Fe4Se4]2+ states with Mössbauer spectroscopy from 1.3 K to 200 K in magnetic fields up to 6.0 T. The spectra of the S- and Se-containing enzymes were found to be remarkably similar. The spectra of the 3-Fe clusters were analyzed and the salient features of the electronic structure are discussed.

Spectroscopic studies of isopenicillin N synthase. A mononuclear nonheme Fe2+ oxidase with metal coordination sites for small molecules and substrate.

The nonheme iron oxidase isopenicillin N synthase catalyzes the formation of two new internal bonds in the tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form the beta-lactam and thiazolidine rings of isopenicillin N. Concomitantly, O2 is reduced to 2 H2O. The recombinant enzyme from Cephalosporium acremonium (Mr = 38,400), expressed as an apoenzyme in Escherichia coli, binds 1 g atom of Fe2+/mol of enzyme to reconstitute full activity. Mössbauer spectra of the 57Fe-enriched enzyme exhibit parameters (delta = 1.30 mm/s, delta EQ = 2.70 mm/s) which unambiguously show that the active site iron is high spin Fe2+. Anaerobic binding of ACV causes a substantial decrease in the isomer shift parameter delta (delta = 1.10 mm/s, delta EQ = 3.40 mm/s) showing that the substrate perturbs the iron site and makes its coordination environment much more covalent. Nitric oxide (NO) binds to the EPR silent active site iron to give an EPR active species (g = 4.09, 3.95, 2.0; S = 3/2) similar to those of the nitrosyl complexes of many other mononuclear Fe2+-containing enzymes. The rhombicity of the EPR spectrum is increased (g = 4.22, 3.81, 1.99) by anaerobic addition of ACV suggesting that the substrate binds to or near the iron without displacing NO. Interestingly, the enzyme.ACV.NO complex displays an optical spectrum similar to that of ferric rubredoxin in which the iron has only thiol coordination. This suggests that the Fe2+ of the enzyme.ACV.NO complex acquires Fe3+ character and that the cysteinyl thiol moiety of ACV coordinates to the iron. Similar substrate thiol coordination to the iron of the enzyme.ACV complex is the most probable explanation for the large decrease in isomer shift observed. These results provide the first evidence for the direct involvement of iron in this unique O2-dependent reaction and suggest novel roles for iron and oxygen in biological catalysis.

Endonuclease III is an iron-sulfur protein.

Elemental analyses, Mössbauer, and EPR data are reported to show that endonuclease III of Escherichia coli is an iron-sulfur protein. Mössbauer spectra of protein freshly prepared from E. coli grown on 57Fe-enriched medium demonstrate that the native enzyme contains a single 4Fe-4S cluster in the 2+ oxidation state, with a net spin of zero. Upon treatment with ferricyanide, a fraction (less than 25%) of the clusters is oxidized into a state which yields an EPR spectrum near g = 2.01 typical of a 3Fe-4S cluster. The magnetic field dependence of the linear electric field effect verifies this assignment. Electron spin echo modulation on the g = 2.01 form of the protein in deuterated solvent indicates the presence of exchangeable protons in the vicinity of the 3Fe-4S cluster. The data obtained show that the [4Fe-4S]2+ cluster of the native enzyme is resistant to either oxidation or reduction, although photoreduction elicited a g = 1.94 type EPR signal characteristic of a [4Fe-4S]1+ cluster. These studies show that endonuclease III is unique in being both a DNA repair enzyme and an iron-sulfur protein. The function of the 4Fe-4S cluster remains to be established.

Evidence for a mu-oxo-bridged binuclear iron cluster in the hydroxylase component of methane monooxygenase. Mössbauer and EPR studies.

Mössbauer and EPR studies of a highly active hydroxylase component of methane monooxygenase isolated from Methylosinus trichosporium OB3b are reported. The Mössbauer spectra of the oxidized (as isolated) hydroxylase show iron in a diamagnetic cluster containing an even number of Fe3+ sites. The parameters are consistent with an antiferromagnetically coupled binuclear cluster similar to those of hemerythrin and purple acid phosphatases. Upon partial reduction of the hydroxylase, an S = 1/2 EPR spectrum with g values at 1.94, 1.86, and 1.75 (gav = 1.85) is observed. Such spectra are characteristic of oxo-bridged iron dimers in the mixed valent Fe(II).Fe(III) state. Further reduction leads to the appearance of a novel EPR resonance at g = 15. Comparison with an inorganic model compound for mu-oxo-bridged binuclear iron suggests that the g = 15 signal is characteristic of the doubly reduced state of the cluster in the protein. In this state, the Mössbauer spectra exhibit two quadrupole doublets typical of high spin Fe2+, consistent with the Fe(II).Fe(II) form of the cluster. The spectral features of the iron center of the hydroxylase in three oxidation states are all similar to those reported for mu-oxo (or mu-hydroxo)-bridged binuclear iron clusters. Since no known monooxygenase contains such a cluster, a new oxygenase mechanism is suggested. Three different preparative methods yielded hydroxylases spanning a 9-fold range in specific activity, yet the same cluster concentration and spectral characteristics were observed. Thus, other parameters than those measured here have a major influence on the activity.

SEM studies of acellular glomerular basement membrane in human diabetic glomerulopathy.

Previous transmission electron microscopic studies have demonstrated glomerular basement membrane (GBM) thickening and mesangial matrix (MM) expansion in chronic stages of diabetes. It is difficult, however, to achieve an appreciation of GBM surface features and distribution of MM in planar views. In the current study, autopsy human renal cortical tissue from patients with end-stage diabetic nephropathy were minced and rendered acellular with detergents prior to fixation, cryofracture, and preparation for light microscopic (LM), transmission electron microscopic (TEM), and scanning electron microscopic (SEM) observation in an effort to visualize extracellular materials in three dimensions. Our studies demonstrated that although diabetic glomerular changes vary widely within and between individuals, most showed alterations primarily affecting peripheral (epithelial) GBM (with MM increased but diffusely distributed), or they exhibited similar GBM changes but with variable nodular MM expansion leading ultimately to capillary occlusion. Both types showed peripheral GBM thickening and demonstrated external surface irregularities that by SEM appeared as "cauliflower-like" lobulations. In these glomeruli, GBM lamellation or reduplication was common with internal layers frequently thrown into lumenward projections. Glomeruli with diffusely distributed MM generally showed patent capillary channels with little evidence of occlusion. By TEM, highly compact, epithelial GBMs were clearly distinguishable from the electron-lucent MM. In these preparations the matrix was concentrated in relatively small discrete masses sometimes covered by a finely fibrillar material, which extended intermittently onto lumenal surfaces of epithelial GBMs. In more advanced stages of MM involvement, glomeruli typically exhibited smooth-surfaced nodules that were increased at the expense of capillary surface area. By TEM, MM nodules were comprised of a meshwork of very fine (20-A) fibrils surrounding a variety of detergent-resistant structures including collagenous fibrils and non-collagenous 30-nm circular fibrils with 16-nm subunits. By SEM, GBM and MM nodules were not distinguishable and merged to form substantial barriers to capillary blood flow. In those capillary channels remaining patent, inwardly projecting folds and ridges were common GBM features, and frequently thin fenestrated layers, distinctly separate from epithelial GBMs, formed sieve-like linings for the channels. These three-dimensional observations provide unique views of the processes leading to diabetic glomerular occlusion and suggest a potential for this technique in the study of renal BM disease.

Ultrastructural analysis of major basement membrane types in rhesus monkey Macaca mulatta acellular renal cortex.

Increasing interest in animal models of human nephropathies have led to a number of renal studies in nonhuman primates. In the current investigation, sequential detergent extraction of cellular elements was carried out on renal cortical tissue blocks from rhesus monkey in an effort to demonstrate clearly the morphological features of major basement membrane (BM) types and their associated extracellular matrix (ECM). LM and TEM views of acellular tissue blocks demonstrate planar arrangements of ECM components, while SEM studies provide striking three-dimensional images of their surface characteristics. All major BM types maintain their in vivo histoarchitectures despite the absence of cells. We propose that the intrinsic structural rigidity of tubular (TBM), Bowman's capsule (BCBM) and peritubular capillary BM (PTCBM) may be related to to their close external association with collagenous fibrils, while glomerular BM (GBM) may be internally supported by a network of mesangial matrix (MM) plates and trabeculae which extend onto internal surfaces of peripheral GBM loops. Thicknesses of rhesus monkey renal BMs show that they are similar to those seen in the laboratory rat and, in general, BCBM greater than TBM greater than GBM greater than PTCBM. We conclude that rhesus monkey renal BMs closely resemble those described by us in the human [J. Ultrastruct. Res. 82: 96-110, 1983] and that this species offers an attractive model for studies of renal diseases of BM origin-notably diabetes mellitus.