NMR characterization of three forms of ferredoxin from Desulphovibrio gigas, a sulphate reducer

A NMR and magnetic susceptibility study of the oxidized and reduced states of three different oligomers (forms) of a [4Fe-4S] ferredoxin protein from Desulphovibrio gigas, FdI, FdI′, and FdII was carried out. FdI and FdI′ are different trimers and FdII a tetramer of the same basic subunit. A probable assignment of the contact shifted resonances is indicated. Since the temperature dependences of the contact shifted resonances associated with each [4Fe-4S] are not all similar a delocalized model for the spin densities on the 4Fe does not apply. The exchange rate between oxidized and reduced states is slow on the NMR time scale. The three oligomers are not magnetically equivalent. Using the “three state hypothesis” terminology it is shown that FdI_ox is predominantly in the C^2? state and changes upon reduction into the C^3? state, while FdII_ox is in the C^? state and changes into the C^2? state. FdI′ does not easily fit into this classification. This study shows a similarity of magnetic behaviour between FdI and bacterial ferredoxins (e.g. Bacillus polymyxa) and between FdII and HiPIP from Chromatium sp.. The influence of the quaternary structure on the stabilization of the different oxidation states of ferredoxins as well as on their redox potentials is discussed.     

NMR redox studies of Desulfovibrio vulgaris Cytochrome c3. Electron transfer mechanisms

The 300-MHz proton NMR spectra of the tetrahaem cytochrome c3 from Desulfovibrio vulgaris were examined while varying the pH and the redox potential. The analysis of the complete NMR reoxidation pattern was done taking into account all the 16 redox states that can be present in the redox titration of a tetra-redox-center molecule. A network of saturation transfer experiments performed at different oxidation stages, between the fully reduced and the fully oxidized states, allowed the observation of different resonances for some of the haem methyl groups. In the present experimental conditions, some of the haems show a fast intramolecular electron exchange rate, but the intermolecular electron exchange is always slow. In intermediate reoxidation stages, large shifts of the resonances of some haem methyl groups were observed upon changing the pH. These shifts are discussed in terms of a pH dependence of the haem midpoint redox potentials. The physiological relevance of this pH dependence is discussed.     

Effect of hydrogen sulfide on growth of sulfate reducing bacteria

A culture of sulfate reducing bacteria (SRB) growing on lactate and sulfate was incubated at different pH values in the range of 5.8-7.0. The effect of pH on growth rate was determined in this pH range; the highest growth rate was observed at pH 6.7. Hydrogen sulfide produced from sulfate reduction was found to have a direct and reversible toxicity effect on the SRB. A hydrogen sulfide Concentration of 547 mg/L (16.1 mM) completely inhibited the culture growth. Comparison between acetic acid and hydrogen sulfide inhibition is presented and the concomitant inhibition kinetics are mathematically described. (c) 1992 John Wiley & Sons, Inc.     

M?ssbauer study of the native, reduced and substrate-reacted Desulfovibrio gigas aldehyde oxido-reductase.

The Desulfovibrio gigas aldehyde-oxido-reductase contains molybdenum and iron-sulfur clusters. M?ssbauer spectroscopy was used to characterize the iron-sulfur clusters. Spectra of the enzyme in its oxidized, partially reduced and benzaldehyde-reacted states were recorded at different temperatures and applied magnetic fields. All the iron atoms in D. gigas aldehyde oxido-reductase are organized as [2Fe-2S] clusters. In the oxidized enzyme, the clusters are diamagnetic and exhibit a single quadrupole doublet with parameters (delta EQ = 0.62 +/- 0.02 mm/s and delta = 0.27 +/- 0.01 mm/s) typical for the [2Fe-2S]2+ state. M?ssbauer spectra of the reduced clusters also show the characteristics of a [2Fe-2S]1+ cluster and can be explained by a spin-coupling model proposed for the [2Fe-2S] cluster where a high-spin ferrous ion (S = 2) is antiferromagnetically coupled to a high-spin ferric ion (S = 5/2) to form a S = 1/2 system. Two ferrous sites with different delta EQ values (3.42 mm/s and 2.93 mm/s at 85 K) are observed for the reduced enzyme, indicating the presence of two types of [2Fe-2S] clusters in the D. gigas enzyme. Taking this observation together with the re-evaluated value of iron content (3.5 +/- 0.1 Fe/molecule), it is concluded that, similar to other Mo-hydroxylases, the D. gigas aldehyde oxido-reductase also contains two spectroscopically distinguishable [2Fe-2S] clusters.     

Reconstitution of sodium channels in large liposomes formed by the addition of acidic phospholipids and freeze-thaw sonication

Phosphatidylcholine (PC) alone or with phosphatidylethanolamine (PE) are sufficient for the reconstitution of Na+ channels in planar lipid bilayers. However, when Na+ channels were first reconstituted into liposomes using the freeze-thaw-sonication method, addition of acidic phospholipids, such as phosphatidylserine (PS), to the neutral phospholipids was necessary to obtain a significant toxin-modulated 22Na uptake. To further investigate the acidic phospholipid effect on reconstitution into liposomes, Na+ channels purified from Electrophorus electricus electrocytes were reconstituted into liposomes of different composition by freeze-thaw sonication and the effect of batrachotoxin and tetrodotoxin on the 22Na flux was measured. The results revealed that, under our experimental conditions, the presence of an acidic phospholipid was also necessary to obtain a significant neurotoxin-modulated 22Na influx. Though neurotoxin-modulated 22Na fluxes have been reported in proteoliposomes made with purified Na+ channels and PC alone, the 22Na fluxes were smaller than those found using lipid mixtures containing acidic phospholipids. Electron microscopy of negatively stained proteoliposomes prepared with PC, PC/PS (1:1 molar ratio), and PS revealed that the acidic phospholipid increases the size of the reconstituted proteoliposomes. The increment in size caused by the acidic phospholipid, due to the associated increase in internal volume for 22Na uptake and in area for Na+ channel incorporation, appears to be responsible for the large neurotoxin-modulated 22Na fluxes observed.     

The active centers of adenylylsulfate reductase from Desulfovibrio gigas. Characterization and spectroscopic studies

In order to utilize sulfate as the terminal electron acceptor, sulfate-reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO4) reductase plays one of the major roles, reducing AdoPSO4 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovibrio gigas. The protein is composed of two non-identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (approximately 400 kDa). It is an iron-sulfur, flavin-containing protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa. Low-temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g = 2.02 and only detectable below 20 K. This signal represented a minor species (0.10-0.25 spins/mol) and showed line broadening in the enzyme isolated from 57Fe-grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe-S center I) observable below 30 K and a concomitant decrease in intensity of the g = 2.02 signal were detected. Effects of chemical reductants (ascorbate, H2/hydrogenase-reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g = 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin-spin-coupled nature (observable below 20 K), indicating the presence of at least two iron-sulfur centers, (centers I and II). M?ssbauer studies on 57Fe-enriched D. gigas AdoPSO4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential less than or equal to 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin-type [4Fe-4S] cluster. Center I exhibits spectra with atypical M?ssbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin-type [4Fe-4S] cluster, suggesting a different structure and/or a distinct cluster-ligand environment.     

Spin-equilibrium and heme-ligand alteration in a high-potential monoheme cytochrome (cytochrome c554) from Achromobacter cycloclastes, a denitrifying organism

A c-type monoheme cytochrome c554 (13 kDa) was isolated from cells of Achromobacter cycloclastes IAM 1013 grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination (low-spin form) coexisting with a minor high-spin form as revealed by the contribution at 630 nm. Magnetic susceptibility measurements support the existence of a small contribution of a high-spin form at all pH values, attaining a minimum at intermediate pH values. The mid-point redox potential determined by visible spectroscopy at pH 7.2 is +150 mV. The pH-dependent spin equilibrum and other relevant structural features were studied by 300-MHz 1H-NMR spectroscopy. In the oxidized form, the 1H-NMR spectrum shows pH dependence with pKa values at 5.0 and 8.9. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c554. Forms I and II predominate at low pH values, and the 1H-NMR spectra reveal heme methyl proton resonances between 40 ppm and 22 ppm. These forms have a methionyl residue as a sixth ligand, and C6 methyl group of the bound methionine was identified in the low-field region of the NMR spectra. Above pH 9.6, form III predominates and the 1H-NMR spectrum is characterized by down-field hyperfine-shifted heme methyl proton resonances between 29 ppm and 22 ppm. Two new resonances are observed at congruent to 66 ppm and 54 ppm, and are taken as indicative of a new type of heme coordination (probably a lysine residue). These pH-dependent features of the 1H-NMR spectra are discussed in terms of the heme environment structure. The chemical shifts of the methyl resonances at different pH values exhibit anti-Curie temperature dependence. In the ferrous state, the 1H-NMR spectrum shows a methyl proton resonance at -3.9 ppm characteristic of methionine axial ligation. The electron-transfer rate between ferric and ferrous forms has been estimated to be smaller than 2 x 10(4) M-1 s-1 at pH 5. EPR spectroscopy was also used to probe the ferric heme environment. A prominent signal at gmax congruent to 3.58 and the overall lineshape of the spectrum indicate an almost axial heme environment.     

Hexaheme nitrite reductase from Desulfovibrio desulfuricans. M?ssbauer and EPR characterization of the heme groups

M?ssbauer and EPR spectroscopy were used to characterize the heme prosthetic groups of the nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774), which is a membrane-bound multiheme cytochrome capable of catalyzing the 6-electron reduction of nitrite to ammonia. At pH 7.6, the as-isolated enzyme exhibited a complex EPR spectrum consisting of a low-spin ferric heme signal at g = 2.96, 2.28, and 1.50 plus several broad resonances indicative of spin-spin interactions among the heme groups. EPR redox titration studies revealed yet another low-spin ferric heme signal at g = 3.2 and 2.14 (the third g value was undetected) and the presence of a high-spin ferric heme. M?ssbauer measurements demonstrated further that this enzyme contained six distinct heme groups: one high-spin (S = 5/2) and five low-spin (S = 1/2) ferric hemes. Characteristic hyperfine parameters for all six hemes were obtained through a detailed analysis of the M?ssbauer spectra. D. desulfuricans nitrite reductase can be reduced by chemical reductants, such as dithionite or reduced methyl viologen, or by hydrogenase under hydrogen atmosphere. Addition of nitrite to the fully reduced enzyme reoxidized all five low-spin hemes to their ferric states. The high-spin heme, however, was found to complex NO, suggesting that the high-spin heme could be the substrate binding site and that NO could be an intermediate present in an enzyme-bound form.     

A universal primer mixture for sequence determination at the 3′ ends of cDNAs

We have devised a universal primer which can be used to sequence the 3′-ends of cloned cDNAs containing a polyA tail. The primer consists of an equimolar mixture of three primers: 20 T nucleotides followed by either an A, C, or G nucleotide (5′→3′). With this primer mixture and the dideoxynucleotide chain termination method, we determined the 3′-terminal sequence of human β-actin cDNA in an Okayama-Berg vector, in four parallel sets of reactions containing either a single primer (T₂₀G, T₂₀C, or T₂₀A) or an equimolar mixture of all three primers. Priming with both T₂₀A and the triple mixture gave clearly readable results that agree with the known sequence of the human β-actin gene, and we have applied this method successfully to several other cDNAs in the Okayama-Berg expression vector. Use of this universal primer mixture facilitates determination of sequences at the 3′-ends of cDNAs while by-passing the polyA tail region.     

Interclonal variation in methylation patterns for expressed and non-expressed genes.

A mass culture of human diploid fibroblasts, and eight clones isolated from that mass culture, were examined for methylation patterns in several regions of DNA. Plasmid-inserted cDNA sequences were used as probes for alpha-hCG, beta-globin, A gamma- and G gamma-globin, and beta- and gamma-actin gene regions. Each probe revealed a different clone-specific pattern of DNA methylation, indicating a striking degree of inter-clonal heterogeneity, for those gene regions which are not normally expressed in diploid fibroblasts (alpha-hCG, gamma-globin and beta-globin). Intra-clonal variation was also evident in many instances, implying that heterogeneity could arise de novo in pure cell clones during serial passage. Thus methylation patterns, in particular for repressed genes, appear to be unstably inherited in these cells, and this instability may lead to random derepression in some cell lineages during mitotic growth.