Rubredoxin reductase of Pseudomonas oleovorans. Structural relationship to other flavoprotein oxidoreductases based on one NAD and two FAD fingerprints

The oxidation of alkanes to alkanols by Pseudomonas oleovorans involves a three-component enzyme system: alkane hydroxylase, rubredoxin and rubredoxin reductase. Alkane hydroxylase and rubredoxin are encoded by the alkBFGHJKL operon, while previous studies indicated that rubredoxin reductase is most likely encoded on the second alk cluster: the alkST operon. In this study we show that alkT encodes the 41 x 10(3) Mr rubredoxin reductase, on the basis of a comparison of the expected amino acid composition of AlkT and the previously established amino acid composition of the purified rubredoxin reductase. The alkT sequence revealed significant similarities between AlkT and several NAD(P)H and FAD-containing reductases and dehydrogenases. All of these enzymes contain two ADP binding sites, which can be recognized by a common beta alpha beta-fold or fingerprint, derived from known structures of cofactor binding enzymes. By means of this amino acid fingerprint we were able to determine that one ADP binding site in rubredoxin reductase (AlkT) is located at the N terminus and is involved in FAD binding, while the second site is located in the middle of the sequence and is involved in the binding of NAD or NADP. In addition, we derived from the sequences of FAD binding reductases a second amino acid fingerprint for FAD binding, and we used this fingerprint to identify a third amino acid sequence in AlkT near the carboxy terminus for binding of the flavin moiety of FAD. On the basis of the known architecture and relative spatial orientations of the NAD and FAD binding sites in related dehydrogenases, a model for part of the tertiary structure of AlkT was developed.     

Repeated structure and possible gene duplications in high potential iron protein and rubredoxin

Summary The three-dimensional structures of bacterial high potential iron protein (HIPIP) and rubredoxin have been searched for repeats to test whether these molecules evolved by independent tandem gene duplications. HIPIP has no structural repeats in spite of the observed repeated pattern in the amino acid sequence fromRhodopseudomonas gelatinosa. Rubredoxin fromClostridium pasteurianum has repeated hairpin loops of ten alpha-carbon atoms on both sides of the active centre iron-sulphur complex, which can be superposed within a root mean square deviation of 0.84 Å by rotating about a local pseudo-dyad axis. The structural repeat matches a weak repeat in the amino acid sequence. It is concluded that the sequence repeats in HIPIP are probably a coincidence but that rubredoxin may have evolved by gene duplication from a dimer of two primitive hairpin loops.     

Ferredoxin and rubredoxin from Butyribacterium methylotrophicum: complete primary structures and construction of phylogenetic trees

Complete amino acid sequences of ferredoxin and rubredoxin from Butyribacterium methylotrophicum, a methylotrophic hetero-acetogen, were determined by combination of protease digestion, Edman degradation, carboxypeptidase digestion, and/or partial acid hydrolysis. The ferredoxin was composed of 55 amino acids with a molecular weight of 5,732 excluding iron and sulfur atoms and showed a typical 2[4Fe-4S]-type ferredoxin sequence with an internal repeat at the 14-23 and 42-51 positions. The rubredoxin was composed of 53 amino acids with a molecular weight of 5,672 excluding iron atom and showed a sequence similar to those of other anaerobic rubredoxins. The sequences were compared to those of corresponding proteins from six different bacteria to construct phylogenetic trees, which showed essentially the same topology. The relationships between the ferredoxin sequences from this bacterium and those of Clostridium thermoaceticum and Methanosarcina barkeri, both of which possess a carbonyl-dependent acetyl-CoA metabolic system, are also discussed.     

Expression of a synthetic gene coding for the amino acid sequence of Clostridium pasteurianum rubredoxin.

A synthetic gene based on the published amino acid sequence for Clostridium pasteurianum rubredoxin was constructed, cloned in Escherichia coli 71/18 and expressed using the T7 RNA polymerase/promoter system in E. coli HMS273. UV/visible spectroscopy and metal analyses indicated that the as-isolated synthetic gene product is a mixture of holo-(i.e. iron-containing) rubredoxin and zinc-substituted rubredoxin, with the latter amounting to approximately 70% of the total rubredoxin. The UV/visible absorption and resonance Raman spectra of the cloned holorubredoxin are characteristic of the native rubredoxin-type iron site. N-terminal amino acid sequencing suggests that the gene product consists of at least three polypeptide species with the initial sequences (approximate relative abundances): Met-Met-Lys-... (63%), blocked (30%) and Met-Lys-... (7%). The blocked portion presumably consists of a mixture of nMet-Met-Lys-... and nMet-Lys-..., where nMet represents an amino-blocked methionine residue.     

Crystallographic study of rubredoxin from the bacterium Desulfovibrio desulfuricans strain 27774

Rubredoxin from Desulfovibrio desulfuricans (strain 27774) has been isolated and crystallized. Preliminary amino acid and crystallographic analyses indicate that this rubredoxin is the smallest rubredoxin isolated so far. The amino acid analysis indicates that the molecule is composed of 45 to 48 residues and contains histidine, which is unusual for rubredoxins from anaerobic bacteria. The X-ray diffraction pattern from these crystals reveals they belong to space group P1 with cell parameters: a = 24.92 A, b = 17.79 A, c = 19.72 A, alpha = 101.0 degrees, beta = 83.4 degrees, gamma = 104.5 degrees. The unit cell volume of 8283 A3 indicates a molecule with molecular weight no greater than 5500 and is consistent with the smaller number of amino acids found in this rubredoxin. The solvent content of this rubredoxin crystal appears to be the lowest observed in crystalline proteins.     

Cloning of genes encoding redox proteins of known amino acid sequence from a library of the Desulfovibrio vulgaris (Hildenborough) genome

A library of 900 recombinant phages has been constructed for the genome of Desulfovibrio vulgaris Hildenborough (1.7 x 10(6) bp) by cloning size-fractionated Sau3A fragments (15-20 kb) into the replacement vector lambda-2001. When a hydrogenase gene probe, a 4.7-kb SalI-EcoRI fragment of known nucleotide sequence, was used to screen the plaque lifted library, 23 positive clones were found, which together span 31 kb of D. vulgaris DNA. To facilitate the cloning of genes with oligodeoxynucleotides as probes, DNA was purified for all clones in the library and spotted on a 16 x 16-cm grid of nitrocellulose. This grid was incubated sequentially to identify lambda clones containing the gene for redox proteins of known amino acid sequence: cytochrome c3 (one 18-mer----four clones), flavodoxin (one 17-mer and one 26-mer----one clone) and rubredoxin (one 44-mer----21 clones). The four cyc-positive clones are also recognized by the rubredoxin oligodeoxynucleotide probe. Restriction mapping defines a 35-kb region of the D. vulgaris chromosome in which the rub and cyc loci are separated by 17.5 kb. The nucleotide sequence of the rubredoxin gene was determined and the deduced amino acid sequence found to agree with that determined in Bruschi [Biochim. Biophys. Acta 434 (1976) 4-17] with the exception of Thr-21 which is found to be encoded by GAC, an Asp codon. A plausible ribosome-binding site precedes the N-terminal initiator methionine residue. Rubredoxin does not have an N-terminal signal sequence which is in agreement with the cytoplasmic location of this redox protein.     

Nucleotide sequence and organization of an H2-uptake gene cluster from Rhizobium leguminosarum bv. viciae containing a rubredoxin-like gene and four additional open reading frames.

The nucleotide sequence of a 3.2 kb region following the hydrogenase structural operon (hupSLCDEF) in the H2-uptake gene cluster from Rhizobium leguminosarum by viciae strain 128C53 has been determined. Five closely linked genes encoding products of 16.3 (HupG), 30.5 (HupH), 8.0 (HupI), 18.4 (HupJ) and 38.7 (HupK) kDa were identified 166 bp downstream from hupF. Transposon insertions into hupG, hupH, hupJ and hupK suppress the H2-oxidizing capability of the wild-type strain. The amino acid sequence deduced from hupI contains two Cys-X-X-Cys motifs, characteristic of rubredoxins, separated by 29 amino acid residues showing strong sequence homology with other bacterial rubredoxins. The amino acid-derived sequence from hupG and hupH showed homology to products from genes hyaE and hyaF of the operon encoding hydrogenase 1 from Escherichia coli, and hupJ and hupK were related to open reading frames identified in Rhodobacter capsulatus and Azotobacter vinelandii hydrogenase gene clusters. An involvement of the hupGHIJK gene cluster in redox reactions related to hydrogenase synthesis or activity is predicted on the basis of the function as electron carrier attributed to rubredoxin.     

The Pseudomonas oleovorans alkBAC operon encodes two structurally related rubredoxins and an aldehyde dehydrogenase

The Pseudomonas oleovorans alkBAC operon encodes seven proteins, of which at least three are involved in alkane hydroxylase (alkBA) and alkanol dehydrogenase (alkC) activities. We have determined the nucleotide sequence of the 2.5-kilobase pair alkA region and analyzed the role of its translation products in alkane oxidation. The alkA region contains three coding sequences, encoding two related rubredoxins (alkF and alkG) of 14- and 18-kDa molecular mass and a 52-kDa aldehyde dehydrogenase (alkH). Deletion analysis indicated that neither the 14-kDa alkF gene product (rubredoxin 1) nor the amino-terminal part of the 18-kDa alkG gene product (rubredoxin 2) is required for alkane hydroxylase activity in vivo. The product of the alkH cistron restores growth of a P. oleovorans aldehyde dehydrogenase mutant on aliphatic alcohols and aldehydes. Its amino acid sequence shows considerable homology to previously characterized aldehyde dehydrogenases from mammalian and fungal origin. The nucleotide composition of the alk genes (47% G + C) differs considerably from the G + C content of the P. oleovorans genome suggesting that the alk regulon may originate from an unrelated organism.     

Solution structure of the two-iron rubredoxin of Pseudomonas oleovorans determined by NMR spectroscopy and solution X-ray scattering and interactions with rubredoxin reductase

Here we provide insights into the molecular structure of the two-iron 19-kDa rubredoxin (AlkG) of Pseudomonas oleovorans using solution-state nuclear magnetic resonance (NMR) and small-angle X-ray scattering studies. Sequence alignment and biochemical studies have suggested that AlkG comprises two rubredoxin folds connected by a linker region of approximately 70 amino acid residues. The C-terminal domain (C-Rb) of this unusual rubredoxin, together with approximately 35 amino acid residues of the predicted linker region, was expressed in Escherichia coli, purified in the one-iron form and the structure of the cadmium-substituted form determined at high-resolution by NMR spectroscopy. The structure shows that the C-Rb domain is similar in fold to the conventional one-iron rubredoxins from other organisms, whereas the linker region does not have any discernible structure. This tandem "flexible-folded" structure of the polypeptide chain derived for the C-Rb protein was confirmed using solution X-ray scattering methods. X-ray scattering studies of AlkG indicated that the 70-amino acid residue linker forms a structured, yet mobile, polypeptide segment connecting the globular N- and C-terminal domains. The X-ray scattering studies also showed that the N-terminal domain (N-Rb) has a molecular conformation similar to that of C-Rb. The restored molecular shape indicates that the folded N-Rb and C-Rb domains of AlkG are noticeably separated, suggesting some domain movement on complex formation with rubredoxin reductase to allow interdomain electron transfer between the metal centers in AlkG. This study demonstrates the advantage of combining X-ray scattering and NMR methods in structural studies of dynamic, multidomain proteins that are not suited to crystallographic analysis. The study forms a structural foundation for functional studies of the interaction and electron-transfer reactions of AlkG with rubredoxin reductase, also reported herein.     

Spectroscopic studies of cobalt and nickel substituted rubredoxin and desulforedoxin.

The single iron site of rubredoxin was replaced by nickel and cobalt. The near-infrared/visible/UV spectra of these metal derivatives show ligand-field transitions and charge-transfer bands which closely resemble those of simple tetrathiolate complexes, indicating a tetrahedral arrangement of the sulfur cysteinyl ligands around the metal core. The 1H NMR spectra of the nickel and cobalt derivatives reveal extremely low-field contact shifted resonances of one proton intensity assigned to beta-CH2 and alpha-CH cysteinyl protons. Other well resolved resonances shifted out of the main protein spectral envelope are also observed and probably arise from contact plus pseudocontact shift mechanisms. Rubredoxins from different sulfate reducers were metal substituted and assignments of aliphatic protons are tentatively proposed, taking advantage of the amino acid sequence homologies. The present data is promising in terms of structural analysis of the coordination sphere of the metal core. It was also shown that replacement of the iron atom of desulforedoxin, a close analogue of rubredoxin, by cobalt and nickel was possible.     

The primary structure of rubrerythrin, a protein with inorganic pyrophosphatase activity from Desulfovibrio vulgaris. Comparison with hemerythrin and rubredoxin.

The complete polypeptide chain of rubrerythrin from the sulfate reducing bacterium Desulfovibrio vulgaris, strain Hildenborough NCIB 8303, was found by protein chemical techniques to consist of 191 residues and to have the amino acid sequence [sequence: see text] The C-terminal part of the protein (position 153----191) shows the typical sequence features of rubredoxin, a protein with a nonheme iron center also present in the same and other Desulfovibrio species. Based on the known three-dimensional structure of D. desulfuricans rubredoxin, we propose that the C-terminal part of rubrerythrin is folded in a similar way and suggest that the deletion of the extra 10 residues is compatible with the same basic rubredoxin-fold. After characterization of the C-terminal region, and in contrast to what could be expected from previously published spectroscopic analyses, the N-terminal region 1-152 of rubrerythrin appears to have no sequence similarity with the eukaryotic protein hemerythrin which is known to contain a binuclear iron center bound by 5 histidine ligands. However, the N-terminal region of rubrerythrin does contain 5 histidine residues but they are differently spaced along the peptide chain. We suggest that at least one of the 3 histidine residues located in the rubredoxin-like center of rubrerythrin may be liganded to one iron atom of the hemerythrin-like center. This paper is the first sequence report of a protein with pyrophosphatase activity although the physiological substrate for the rubrerythrin may be not inorganic pyrophosphate.     

The structure of rubredoxin from Desulfovibrio desulfuricans strain 27774 at 1.5 A resolution

The structure of a small rubredoxin from the bacterium Desulfovibrio desulfuricans has been determined and refined at 1.5 A resolution. The hairpin loop containing seven residues in other rubredoxins is missing in this 45 residue molecule, and once that fact was determined by amino acid sequencing studies, refinement progressed smoothly to an R value of 0.093 for all reflections from 5 to 1.5 A resolution. Nearly all of the water molecules in the well-ordered triclinic unit cell have been added to the crystallographic model. As in the other refined rubredoxin models, the Fe-S4 complex is slightly distorted from ideal tetrahedral coordination.     

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.     

Calculation of protein tertiary structure

We describe a method for calculating the tertiary structure of proteins given their amino acid sequence. The algorithm involves locally minimizing an energylike expression as a function of the Cartesian co-ordinates of the C_β of all residues. Although the approximation to the true polypeptide geometry and conformational energies is extremely approximate, quite respectable results have been obtained for the small proteins rubredoxin and trypsin inhibitor, where the root mean square errors are as low as 4.0 Å and 4.7 Å, respectively.     

The biosynthesis of phosphorylated tyrosine hydroxylase by organ cultures of rat adrenal medulla and superior cervical ganglia: a correction.

Determination of the complete amino acid sequence of the rubredoxin isolated from the sulfate reducing bacterium $\text{Desulfovibrio}$$\text{gigas}$ showed that the molecule consists of a single polypeptide chain of 52 residues. The sequence of the first 42 residues was determined using an automatic Protein Sequencer. Peptides derived from tryptic hydrolysis and from specific cleavage at tryptophan residue were used to construct the total sequence. Compared with the sequence of $\text{Desulfovibrio}$$\text{vulgaris}$ rubredoxin, 37 positions are identical, and with the sequences of $\text{Clostridium}$$\text{pasteurianum}$, $\text{Peptostreptococcus}$$\text{elsdenii}$, $\text{Micrococcus}$$\text{aerogenes}$ and $\text{D.}$$\text{vulgaris}$ rubredoxins, 20 matching residues occur. A crystallographic study of the $\text{D.}$$\text{gigas}$ rubredoxin is in progress.     

Isolation and characterization of a rubredoxin and an (8Fe-8S) ferredoxin from Desulfuromonas acetoxidans

A two cluster (4Fe-4S) ferredoxin and a rubredoxin have been isolated from the sulfur-reducing bacterium Desulfuromonas acetoxidans. Their amino acid compositions are reported and compared to those of other iron-sulfur proteins. The ferredoxin contains 8 cysteine residues, 8 atoms of iron and 8 atoms of labile sulfur per molecule; its minimum molecular weight is 6163. The protein exhibits an abosrbance ratio of A385/A283 = 0.74. Storage results in a bleaching of the chromophore; the denatured ferredoxin is reconstitutable with iron and sulfide. The instability temperature is 52 degrees C. The rubredoxin does not differ markedly from rubredoxins from other anaerobic bacteria.     

Distribution of the Rubredoxin Gene Among the Clostridium butyricum Species

With PCR methods, the rubredoxin gene was systematically identified among 11 strains of Clostridium butyricum; this ubiquity means major functions in the metabolism of the Clostridia. The 11 PCR products allowed deduction of a sequence of 26 amino acids corresponding to positions 11–36 of the rubredoxin. They all contained the tyrosines at positions 11 and 13 and the phenylalanine at position 30 characteristic of the rubredoxin, but differed at positions 14–17, 20, 25, 29, and 31, allowing determination of three types of rubredoxins among these 11 strains of C. butyricum. Received: 13 October 1998 / Accepted: 23 November 1998