Iron-sulfur clusters of biotin synthase in vivo: a Mössbauer study.

Biotin synthase, the enzyme that catalyzes the last step of the biosynthesis of biotin, contains only [2Fe-2S](2+) clusters when isolated under aerobic conditions. Previous results showed that reconstitution with an excess of FeCl(3) and Na(2)S under reducing and anaerobic conditions leads to either [4Fe-4S](2+), [4Fe-4S](+), or a mixture of [4Fe-4S](2+) and [2Fe-2S](2+) clusters. To determine whether any of these possibilities or other different cluster configuration could correspond to the physiological in vivo state, we have used (57)Fe Mössbauer spectroscopy to investigate the clusters of biotin synthase in whole cells. The results show that, in aerobically grown cells, biotin synthase contains a mixture of [4Fe-4S](2+) and [2Fe-2S](2+) clusters. A mixed [4Fe-4S](2+):[2Fe-2S](2+) cluster form has already been observed under certain in vitro conditions, and it has been proposed that both clusters might each play a significant role in the mechanism of biotin synthase. Their presence in vivo is now another argument in favor of this mixed cluster form.

Fate of the (2Fe-2S)(2+) cluster of Escherichia coli biotin synthase during reaction: a Mössbauer characterization.

Biotin synthase, the enzyme which catalyzes the last step of the biosynthesis of biotin, contains only (2Fe-2S)(2+) clusters when isolated under aerobic conditions. Previous results showed that reduction by dithionite or photoreduced deazaflavin converts the (2Fe-2S)(2+) to (4Fe-4S)(2+,+). However, until now, no detailed investigation concerning the fate of the (2Fe-2S)(2+) during reduction under assay conditions (NADPH, flavodoxin, flavodoxin reductase) has been realized. Here, we show by Mössbauer spectroscopy on a partially purified fraction overexpressing the enzyme that, in the presence of a S(2)(-) source and Fe(2+), there is conversion of the predominant (2Fe-2S)(2+) clusters into a 1:1 mixture of (2Fe-2S)(2+) and (4Fe-4S)(2+). No change in this cluster composition was observed in the presence of the physiological reducing system. When the reaction was allowed to proceed by addition of the substrate dethiobiotin, the (4Fe-4S)(2+) was untouched whereas the (2Fe-2S)(2+) was degraded into a new species. This is consistent with the hypothesis that the reduced (4Fe-4S) cluster is involved in mediating the cleavage of AdoMet and that the (2Fe-2S)(2+) is the sulfur source for biotin.

Metastable isonitrosyl structure of the nitroprusside anion confirmed by nuclear inelastic scattering.

Nuclear inelastic scattering (NIS) measurements were performed on a guanidium nitroprusside ((CN(3)H(6))(2)[Fe(CN)(5)NO], GNP) monocrystal at 77 K after the sample was illuminated with blue light (450 nm) at 50 K to populate the two metastable states, MS(1) and MS(2), of the nitroprusside anion. A second measurement was performed at 77 K after warming up the illuminated crystal to 250 K where the metastable states decay to the groundstate. The measured spectra were compared with simulated NIS spectra that were calculated by using density functional methods. Comparison of measured and simulated spectra provides strong evidence for the isonitrosyl structure of the metastable MS(1) state proposed by Carducci et al. (Carducci, M. D.; Pressprich, M. R.; Coppens, P. J. Am. Chem. Soc. 1997, 119, 2669-2678).

The presence of an iron-sulfur cluster in adenosine 5'-phosphosulfate reductase separates organisms utilizing adenosine 5'-phosphosulfate and phosphoadenosine 5'-phosphosulfate for sulfate assimilation.

It was generally accepted that plants, algae, and phototrophic bacteria use adenosine 5'-phosphosulfate (APS) for assimilatory sulfate reduction, whereas bacteria and fungi use phosphoadenosine 5'-phosphosulfate (PAPS). The corresponding enzymes, APS and PAPS reductase, share 25-30% identical amino acids. Phylogenetic analysis of APS and PAPS reductase amino acid sequences from different organisms, which were retrieved from the GenBank(TM), revealed two clusters. The first cluster comprised known PAPS reductases from enteric bacteria, cyanobacteria, and yeast. On the other hand, plant APS reductase sequences were clustered together with many bacterial ones, including those from Pseudomonas and Rhizobium. The gene for APS reductase cloned from the APS-reducing cyanobacterium Plectonema also clustered together with the plant sequences, confirming that the two classes of sequences represent PAPS and APS reductases, respectively. Compared with the PAPS reductase, all sequences of the APS reductase cluster contained two additional cysteine pairs homologous to the cysteine residues involved in binding an iron-sulfur cluster in plants. Mössbauer analysis revealed that the recombinant APS reductase from Pseudomonas aeruginosa contains a [4Fe-4S] cluster with the same characteristics as the plant enzyme. We conclude, therefore, that the presence of an iron-sulfur cluster determines the APS specificity of the sulfate-reducing enzymes and thus separates the APS- and PAPS-dependent assimilatory sulfate reduction pathways.

Models of the bis-histidine-coordinated ferricytochromes: Mössbauer and EPR spectroscopic studies of low-spin iron(III) tetrapyrroles of various electronic ground states and axial ligand orientations.

The EPR and magnetic Mössbauer spectra of a series of axial ligand complexes of tetrakis(2,6-dimethoxyphenyl)porphyrinatoiron(III), [(2,6-(OMe)(2))(4)TPPFeL(2)](+), where L= N-methylimidazole, 2-methylimidazole, or 4-(dimethylamino)pyridine, of one axial ligand complex of tetraphenylporphyrin, the bis(4-cyanopyridine) complex [TPPFe(4-CNPy)(2)](+), and of one axial ligand complex of tetraphenylchlorin, [TPCFe(ImH)(2)](+), where ImH=imidazole, have been investigated and compared to those of low-spin Fe(III) porphyrinates and ferriheme proteins reported in the literature. On the basis of this and previous complementary spectroscopic investigations, three types of complexes have been identified: those having (d(xy))(2)(d(xz),d(yz))(3) electronic ground states with axial ligands aligned in perpendicular planes (Type I), those having (d(xy))(2)(d(xz),d(yz))(3) electronic ground states with axial ligands aligned in parallel planes (Type II), and those having the novel (d(xz),d(yz))(4)(d(xy))(1) electronic ground state (Type III). A subset of the latter type, with planar axial ligands aligned parallel to each other or strong macrocycle asymmetry that yield rhombic EPR spectra, cannot be created using the porphyrinate ligand. Type I centers are characterized by "large g(max)" EPR spectra with g>3.2 and well-resolved, widely spread magnetic Mössbauer spectra having A(zz)/ g(N)mu(N)>680 kG, with A(xx) negative in sign but much smaller in magnitude than A(zz), while Type II centers have well-resolved rhombic EPR spectra with g(zz)=2.4-3.1 and also less-resolved magnetic Mössbauer spectra, and usually have A(zz)/ g(Nmu(N) in the range of 440-660 kG (but in certain cases as small as 180 kG) and A(xx) again negative in sign but only somewhat smaller (but occasionally larger in magnitude) than A(zz), and Type III centers have axial EPR spectra with g( upper left and right quadrants ) approximately 2.6 or smaller and g( vertical line )<1.0-1.95, but often not resolved, and less-resolved magnetic Mössbauer spectra having A(zz)/ g(N)mu(N) in the range of 270-400 kG, and A(xx) again negative in sign but much smaller in magnitude than A(zz). An exception to this rule is [TPPFe(4-CNPy)(2)](+), which has A(xx)/ g(N)mu(N)=-565 kG, A(yy)/ g(N)mu(N)=629 kG, and A(zz)/ g(N)mu(N)=4 kG. A subset of Type II complexes (Type II') have rhombicities ( V/Delta) much greater than 0.67 and A(zz)/ g(N)mu(N) ranging from 320 to 170 kG, with A(xx) also negative but with the magnitude of A(xx) significantly larger than that of A(zz). These classifications are also observed for a variety of ferriheme proteins, and they lead to linear correlations between A(zz) and either A(xx), g(zz), or V/Delta for Types I and II (but not for A(zz) versus V/Delta for Type II'). Not enough data are yet available on Type III complexes to determine what, if any, correlations may be observed.