Search

Dicamba O -demethylase is a multicomponent enzyme that catalyzes the conversion of the herbicide 2-methoxy-3,6-dichlorobenzoic acid (dicamba) to 3,6-dichlorosalicylic acid (DCSA). The three components of the enzyme were purified and characterized. Oxygenase DIC is a homotrimer (α) 3 with a subunit molecular mass of approximately 40 kDa. Ferredoxin DIC and reductase DIC are monomers with molecular weights of approximately 14 and 45 kDa, respectively. EPR spectroscopic analysis suggested the presence of a single [2Fe–2S] (2+/1+) cluster in ferredoxin DIC and a single Rieske [2Fe–2S] (2+; 1+) cluster within oxygenase DIC . Consistent with the presence of a Rieske iron–sulfur cluster, oxygenase DIC displayed a high reduction potential of E m,7.0 = −21 mV whereas ferredoxin DIC exhibited a reduction potential of approximately E m,7.0 = −171 mV. Optimal oxygenase DIC activity in vitro depended on the addition of Fe 2+ . The identification of formaldehyde and DCSA as reaction products demonstrated that dicamba O -demethylase acts as a monooxygenase. Taken together, these data suggest that oxygenase DIC is an important new member of the Rieske non-heme iron family of oxygenases.

Iron clusters are the catalytically active groups of many proteins. The basis building blocks of these clusters are few; they are all variants of three archetypal patterns: [Fe–O–Fe], [2Fe–2S], and [4Fe–4S]. Mossbauer spectroscopy, in conjunction with EPR spectroscopy, susceptometry and EXAFS, plays an important role in characterizing type, number, oxidation states and magnetic properties of such clusters in newly discovered proteins, and in detecting how valency and magnetism vary by passing through the catalytical cycle of these proteins. In this contribution we present the characterization of a novel iron cluster with unusual bridging and terminal ligands and metal coordinations.

Aldehyde:ferredoxin oxidoreductase (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus is a homodimeric protein. Each subunit carries one [4Fe-4S] cubane and a novel tungsten cofactor containing two pterins. A single iron atom bridges between the subunits. AOR has previously been studied with EPR spectroscopy in an inactive form known as the red tungsten protein (RTP): reduced RTP exhibits complex EPR interaction signals. We have now investigated the active enzyme AOR with EPR, and we have found an S = 1/2 plus S = 3/2 spin mixture from a non-interacting [4Fe-4S]1+ cluster in the reduced enzyme. Oxidized AOR affords EPR signals typical for W(V) with g–values of 1.982, 1.953, and 1.885. The W(V) signals disappear at a reduction potential E m,7.5 of +180 mV. This unexpectedly high value indicates that the active-site redox chemistry is based on the pterin part of the cofactor.

The dissimilatory sulfite reductase desulfoviridin was purified from the membrane (mSiR) and the soluble fraction (sSiR) of the sulfate-reducing bacterium Desulfovibrio desulfuricans (Essex). Molecular and spectroscopic properties were determined and compared with the properties of the soluble desulfoviridin from Desulfovibrio vulgaris (Hildenborough). The enzymes were isolated as alpha 2 beta 2 gamma n (n = 1-3) multimers with a relative molecular mass of 200 +/- 10 (gel filtration). Both mSiR and sSiR from D. desulfuricans contained 24 +/- 3 Fe and 18 +/- 3 labile sulfide/200 kDa, respectively, and showed identical EPR spectra. Quantification of the high-spin Fe(III) heme resonances at g of approximately 6 indicated that close to 80% of the siroheme moiety in the enzyme from D. desulfuricans was demetallated. D. desulfuricans sulfite reductase showed S = 9/2 EPR signals with the highest apparent g value at g = 17 as reported for SiR from D. vulgaris. Antibodies raised against the alpha, beta and gamma subunit of the D. vulgaris enzyme exhibited cross-reactivity with the subunits of mSiR and sSiR from D. desulfuricans. N-terminal sequences of alpha, beta and gamma subunits of both mSiR and sSiR from D. desulfuricans were identical and showed a high degree of similarity with the sequences of the corresponding subunits obtained from the D. vulgaris enzyme. During gel filtration of sSiR from D. desulfuricans, under non-denaturing conditions, a small protein (molecular mass approximately 11 kDa) was separated. This 11-kDa protein exhibited cross-reactivity with the antibody raised against the gamma subunit of D. vulgaris sulfite reductase. In the case of D. desulfuricans sulfite reductase, the 11-kDa gamma subunit seems not to be an integral part of the protein and can be obtained from the soluble fraction and during purification of the soluble enzyme.

The purification and characterization of a ferredoxin from Desulfovibrio vulgaris (Hildenborough) is described. The protein can be isolated in two forms; the major form is strongly complexed to RNA, while a minor form is free from nucleic acid. Bound RNA cannot be removed by digestion with nucleases, or by heating to 70 °C, and it can only be partially removed by rechromatography. The ultraviolet/visible spectrum shows typical absorption maxima at 280 nm and 400 nm for the RNA-free ferredoxin. The RNA-bound protein exhibits an additional strong peak at 260 nm. The RNA can be extracted from the protein with phenol. The ferredoxin is a dimer of subunits, each of 7.5 kDa; its pI is 3.9. The protein contains a [4Fe–4S](2+;1+) cluster with an EPR spectrum (g= 1.90, 1.93 and 2.05) in the reduced state. A reduction potential of – 360 mV was determined for the RNA-free ferredoxin with reversible voltammetry at glassy carbon. From the temperature dependence of the reduction potential, the unusually high standard reaction entropy was calculated as ?S˚=– 230 J · K-1· mol-1. No electrochemical response was obtained with the RNA-bound ferredoxin. Binding of RNA appears to require the presence of an intact cluster, since the absence of absorption at 400 nm runs in parallel with the absence of absorption at 260 nm. The possibility is discussed that the binding to the RNA has a regulatory function and is controlled by the state of the cluster.

Nitrate has been shown to shunt the electron flow in Clostridium thermoaceticum from CO 2 to nitrate, but it did not influence the levels of enzymes involved in the Wood-Ljungdahl pathway (J. M. Fröstl, C. Seifritz, and H. L. Drake, J. Bacteriol. 178:4597–4603, 1996). Here we show that under some growth conditions, nitrate does in fact repress proteins involved in the Wood-Ljungdahl pathway. The CO oxidation activity in crude extracts of nitrate (30 mM)–supplemented cultures was fivefold less than that of nitrate-free cultures, while the H 2 oxidation activity was six- to sevenfold lower. The decrease in CO oxidation activity paralleled a decrease in CO dehydrogenase (CODH) protein level, as confirmed by Western blot analysis. Protein levels of CODH in nitrate-supplemented cultures were 50% lower than those in nitrate-free cultures. Western blots analyses showed that nitrate also decreased the levels of the corrinoid iron-sulfur protein (60%) and methyltransferase (70%). Surprisingly, the decrease in activity and protein levels upon nitrate supplementation was observed only when cultures were continuously sparged. Northern blot analysis indicates that the regulation of the proteins involved in the Wood-Ljungdahl pathway by nitrate is at the transcriptional level. At least a 10-fold decrease in levels of cytochrome b was observed with nitrate supplementation whether the cultures were sparged or stoppered. We also detected nitrate-inducible nitrate reductase activity (2 to 39 nmol min −1 mg −1 ) in crude extracts of C. thermoaceticum . Our results indicate that nitrate coordinately represses genes encoding enzymes and electron transport proteins in the Wood-Ljungdahl pathway and activates transcription of nitrate respiratory proteins. CO 2 also appears to induce expression of the Wood-Ljungdahl pathway genes and repress nitrate reductase activity.

The prismane protein of Desulfovibrio vulgaris, in its isolated, its one-electron-reduced and its oxidized states, was the subject of a detailed Mossbauer investigation. Measurements were recorded in the range 0.295-77 K and in the field range 0-6.2 T (parallel and perpendicular to the gamma beam). The paramagnetic parts of the magnetically split Mossbauer spectra were analyzed with the spin-Hamiltonian formalism, including the nuclear Hamiltonian; the diamagnetic parts result from the nuclear Hamiltonian only. The field-dependent spectra at 295 mK and 4.2 K indicate that the paramagnetic part of the isolated protein represents a spin-coupled 4Fe unit with the spin of one Fe site (5/2) oriented antiparallel to the spins of the other three Fe sites (5/2, 5/2 and 2), yielding a total cluster spin, Stot of 9/2. The Mossbauer parameters of the individual Fe sites indicated that this unit represents a 4Fe cluster with an unusual mixture of bridging and terminal ligands and metal coordinations (hybrid cluster). The diamagnetic part of the isolated protein represents an additional 4Fe unit, which, according to its Mossbauer parameters, is a [4Fe2.5+-4S] cubane. The parameter changes upon one-electron oxidation or reduction and the magnetic properties of the two clusters in the three oxidation states of the protein investigated here reveal that the redox behavior of the prismane protein is exclusively related with the hybrid cluster. [structures: see text] These findings are contrary to the former hypothesis of one or two [6Fe-6S] cluster(s) as the prosthetic group of this protein [Hagen, W. R., Pierik, A. J. & Veeger, C. (1989) J. Chem. Soc. Faraday Trans. 185, 4083-4090; Moura, I., Tavares, P., Moura, J. J. G., Ravi, N., Huynh, B. H., Liu, M.-Y. & LeGall, J. (1992) J. Biol. Chem. 287, 4487-4496]. However, they are in full agreement with the crystal structure of the isolated protein, which, concurrent with our Mossbauer investigation, has been solved.

Crystals of the prismane protein from Desulfovibrio vulgaris (Hildenborough) containing a putative [6Fe-6S] cluster have been obtained and X-ray data collected to a resolution of 1.7 A using synchrotron radiation. The unit cell is orthorhombic with a = 64.1, b = 65.1 and c = 154.1 A, space group P2(1)2(1)2(1) (No. 19). The unit cell will readily accommodate four molecules of molecular weight 60 kDa with a corresponding solvent content of approximately 48%.

Three groups that absorb in the 2100-1800-cm-1 infrared spectral region have recently been detected in Ni-hydrogenase from Chromatium vinosum [Bagley, K.A., Duin, E.C., Roseboom, W., Albracht, S. P.J. & Woodruff, W.H. (1995) Biochemistry 34, 5527-5535]. To assess the significance and generality of this observation, we have carried out an infrared-spectroscopic study of eight hydrogenases of three different types (nickel, iron and metal-free) and of 11 other iron-sulfur and/or nickel proteins. Infrared bands in the 2100-1800-cm-1 spectral region were found in spectra of all Ni-hydrogenases and Fe-hydrogenases and were absent from spectra of any of the other proteins, including a metal-free hydrogenase. The positions of these bands are dependent on the redox state of the hydrogenase. The three groups in Ni-hydrogenases that are detected by infrared spectroscopy are assigned to the three unidentified small non-protein ligands that coordinate iron in the dinuclear Ni/Fe active site as observed in the X-ray structure of the enzyme from Desulfovibrio gigas [Volbeda, A., Charon, M.-H., Piras, C., Hatchikian, E.C., Frey, M. & Fontecilla-Camps, J.C. (1995) Nature 373, 580-587]. It is concluded that these groups occur exclusively in metal-containing H2-activating enzymes. It is proposed that the active sites of Ni-hydrogenases and of Fe-hydrogenases have a similar architecture, that is required for the activation of molecular hydrogen.

Fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 130-fold under anoxic conditions. The native enzyme had an apparent molecular mass of 114 kDa and was composed of two subunits of 60 kDa. The enzyme exhibited maximum activity at pH 8.5 and approximately 54° C. The K m values for fumarate and l-malate were 0.25 mM and 2.38 mM, respectively. Fumarase was inactivated by oxygen, but the activity could be restored by addition of Fe2+ and β-mercaptoethanol under anoxic conditions. EPR spectroscopy of the purified enzyme revealed the presence of a [3Fe-4S] cluster. Under reducing conditions, only a trace amount of a [4Fe-4S] cluster was detected. Addition of fumarate resulted in a significant increase of this [4Fe-4S] signal. The N-terminal amino acid sequence showed similarity to the sequences of fumarase A and B of Escherichia coli (56%) and fumarase A of Salmonella typhimurium (63%).

The sulfhydrogenase from the extreme thermophile Pyrococcus furiosus has been re-investigated. The αβγδ heterotetrameric enzyme of 153.3 kDa was found to contain 17 Fe, 17 S2−, and 0.74 Ni. The specific activity of the purified protein was 80 U/mg. Three EPR signals were found. A rhombic S = 1 2 signal (g = 2.07, 1.93, 1.89) was observed reminiscent in its shape and temperature dependence of spectra from [4Fe-4S](2+;1+) clusters. However, in reductive titrations the spectrum appeared at the unusually high potential Em,7.5 = −90 mV. Moreover, the signal dissappeared again at Em,7.5 = −328 mV. Also, two other signals appear upon reduction: a near-axial (g = 2.02, 1.95, 1.92) S = 1 2 spectrum (Em,7.5 = −303 mV) indicative for the presence of a [2Fe-2S](2+;1+) cluster, and a broad spectrum of unknown origin with effective g-values 2.25, 1.89 (Em,7.5 = −310 mV). We hypothesize that the latter signal is caused by magnetic interaction of the rhombic signal and a third cluster.

The active site of Escherichia coli NADPH-sulfite reductase has previously been modeled as a siroheme with its iron bridged to a nearby iron-sulfur cubane, resulting in antiferromagnetic exchange coupling between all iron atoms. The model has been suggested to hold also for other sulfite reductases and nitrite reductases. We have recently challenged the generality of the model with the finding that the EPR of Fe/S in dissimilatory sulfite reductase (desulfoviridin) from Desulfovibrio vulgaris indicates that an S = 9/2 system is not subject to coupling. Siroheme in desulfoviridin is to a large extent demetalated, and therefore coupling is physically impossible. We have now studied examples from a second class of dissimilatory sulfite reductases, desulforubidins, which have their siroporphyrins fully metalated. Desulforubidin from Desulfosarcina variabilis is a 208-kDa alpha 2 beta 2 gamma 2 hexamer. The alpha- and beta-subunits are immunologically active with antibodies raised against the corresponding subunits from D. vulgaris desulfoviridin, whereas the gamma-subunit is not. The desulforubidin contains two fully metalated sirohemes and a total of approximately 15 Fe and approximately 19 S2-. Quantification of high-spin plus low-spin heme EPR signals accounts for all sirohydrochlorin. The frequency-independent (9-35 GHz) effective perpendicular g-values of the high-spin S = 5/2 siroheme (6.33, 5.19) point to quantum mixing with an excited (approximately 770 cm-1) S = 3/2 multiplet. Similar anomalous g-values are observed with sulfite reductases from Desulfovibrio baarsii and Desulfotomaculum acetoxidans. The D. variabilis enzyme exhibits very approximately stoichiometric S = 9/2 EPR (g = 16).(ABSTRACT TRUNCATED AT 250 WORDS)

Nitrogenase is a two-component metalloenzyme that catalyzes a MgATP hydrolysis driven reduction of substrates. Aluminum fluoride plus MgADP inhibits nitrogenase by stabilizing an intermediate of the on-enzyme MgATP hydrolysis reaction. We report here the redox properties and electron paramagnetic resonance (EPR) signals of the aluminum fluoride-MgADP stabilized nitrogenase complex of Azotobacter vinelandii. Complex formation lowers the midpoint potential of the [4Fe-4S] cluster in the Fe protein. Also, the two-electron reaction of the unique [8Fe-7S] cluster in the MoFe protein is split in two one-electron reactions both with lower midpoint potentials. Furthermore, a change in spin-state of the two-electron oxidized [8Fe-7S] cluster is observed. The implications of these findings for the mechanism of MgATP hydrolysis driven electron transport within the nitrogenase protein complex are discussed.