Hyperfine-shifted (13)C and (15)N NMR signals from Clostridium pasteurianum rubredoxin: extensive assignments and quantum chemical verification.

Stable isotope-labeling methods, coupled with novel techniques for detecting fast-relaxing NMR signals, now permit detailed investigations of paramagnetic centers of metalloproteins. We have utilized these advances to carry out comprehensive assignments of the hyperfine-shifted (13)C and (15)N signals of the rubredoxin from Clostridium pasteurianum (CpRd) in both its oxidized and reduced states. We used residue-specific labeling (by chemical synthesis) and residue-type-selective labeling (by biosynthesis) to assign signals detected by one-dimensional (15)N NMR spectroscopy, to nitrogen atoms near the iron center. We refined and extended these (15)N assignments to the adjacent carbonyl carbons by means of one-dimensional (13)C[(15)N] decoupling difference experiments. We collected paramagnetic-optimized SuperWEFT (13)C[(13)C] constant time COSY (SW-CT-COSY) data to complete the assignment of (13)C signals of reduced CpRd. By following these (13)C signals as the protein was gradually oxidized, we transferred these assignments to carbons in the oxidized state. We have compared these assignments with hyperfine chemical shifts calculated from available X-ray structures of CpRd in its oxidized and reduced forms. The results allow the evaluation of the X-ray structural models as representative of the solution structure of the protein, and they provide a framework for future investigation of the active site of this protein. The methods developed here should be applicable to other proteins that contain a paramagnetic center with high spin and slow electron exchange.

Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain.

We have identified a novel family of proteins, in which the N-terminal cystathionine beta-synthase (CBS) domain is fused to the C-terminal Zn ribbon domain. Four proteins were overexpressed in Escherichia coli and purified: TA0289 from Thermoplasma acidophilum, TV1335 from Thermoplasma volcanium, PF1953 from Pyrococcus furiosus, and PH0267 from Pyrococcus horikoshii. The purified proteins had a red/purple color in solution and an absorption spectrum typical of rubredoxins (Rds). Metal analysis of purified proteins revealed the presence of several metals, with iron and zinc being the most abundant metals (2-67% of iron and 12-74% of zinc). Crystal structures of both mercury- and iron-bound TA0289 (1.5-2.0 A resolution) revealed a dimeric protein whose intersubunit contacts are formed exclusively by the alpha-helices of two cystathionine beta-synthase subdomains, whereas the C-terminal domain has a classical Zn ribbon planar architecture. All proteins were reversibly reduced by chemical reductants (ascorbate or dithionite) or by the general Rd reductase NorW from E. coli in the presence of NADH. Reduced TA0289 was found to be capable of transferring electrons to cytochrome C from horse heart. Likewise, the purified Zn ribbon protein KTI11 from Saccharomyces cerevisiae had a purple color in solution and an Rd-like absorption spectrum, contained both iron and zinc, and was reduced by the Rd reductase NorW from E. coli. Thus, recombinant Zn ribbon domains from archaea and yeast demonstrate an Rd-like electron carrier activity in vitro. We suggest that, in vivo, some Zn ribbon domains might also bind iron and therefore possess an electron carrier activity, adding another physiological role to this large family of important proteins.

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.

Rubredoxin from Clostridium perfringens: complete amino acid sequence and participation in nitrate reduction.

The complete primary structure of rubredoxin (Rd) isolated from Clostridium perfringens was sequenced to be: MKKFICDVCGYIYDPAVGDPDNGVEPGTEFKDIPDDWVCPLCGVDKSQFSETEE. The sequence was highly homologous to that of C. pasteurianum Rd but was different at 13 sites out of the total 54 amino acid residues (76% homology). It contained 1 Fe atom, 4 cysteine residues, and no labile sulfur, had a molecular weight of 6,056, and shared the general properties of classical anaerobic Rds. The pI was 4.4. The Rd was reduced with NADH in the presence of a specific NAD(P)H oxidoreductase preparation from the bacterium. The Km value of nitrate reductase for Rd as an electron-donor was 12 microM, a value comparable to that of the 13 microM for ferredoxin (Fd). These results taken together provide additional support for its role as the electron carrier in the nitrate reductase system [Seki, S., Ikeda, A., and Ishimoto, M. (1988) J. Biochem. 103, 583-584].

Crystallization and preliminary X-ray diffraction study of a protein with a high potential rubredoxin center and a hemerythrin-type Fe center.

A newly discovered iron-containing protein, isolated from the bacterium Desulfovibrio vulgaris (Hildenborough, NCIB 8303), has been crystallized. The molecule appears to be a dimer of mass 44kDa. This protein has iron centers with spectrascopic similarities to those in rubredoxins and in hemerythrins. The X-ray diffraction shows symmetry consistent with space group I222 or I212121. Cell parameters are a = 49.2 A, b = 81.3 A, c = 100.1 A, and alpha, beta, gamma = 90 degrees. X-ray diffraction data have been collected to 3.0 A, and a search for useful heavy atom derivatives is in progress for the analysis of the crystal structure of this Fe-protein.

Differential levels of ferredoxin and rubredoxin in Clostridium acetobutylicum.

Ferredoxin and rubredoxin levels have been determined in DEAE-cellulose treated extracts of Clostridium acetobutylicum using specific enzymatic assays. In contrast to ferredoxin, the content of rubredoxin is affected by various culture conditions; it fluctuates in the proportions of 1 to 3 according to the growth phase, 1 to 8 according to the medium composition, and 1 to 40 according to the pH. Highest rubredoxin level is obtained at the end of the acid phase when the cells grow in chemically defined medium, the pH of which is not controlled. Such variations suggest that rubredoxin as well as rubredoxin-reductase take part in an electron transport chain system inducible by the culture conditions.

Characterization of ferredoxin, flavodoxin, and rubredoxin from Clostridium formicoaceticum grown in media with high and low iron contents.

Ferredoxin, flavodoxin, and rubredoxin were purified to homogeneity from Clostridium formicoaceticum and characterized. Variation of the iron concentration of the growth medium caused substantial changes in the concentrations of ferredoxin and flavodoxin but not of rubredoxin. The ferredoxin has a molecular weight of 6,000 and is a four iron-four sulfur protein with eight cysteine residues. The spectrum is similar to that of other ferredoxins. The molar extinction coefficients are 22.6 X 10(3) and 17.6 X 10(3) at 280 and 390 nm, respectively. From 100 g wet weight of cells grown with 3.6 microM iron and with 40 microM iron, 5 and 20 mg offerredoxin were isolated, respectively. The molecular weight of rubredoxin is 5,800 and it contains one iron and four cysteines. The UV-visible absorption spectrum is dissimilar to those of other rubredoxins in that the 373 nm absorption peak is quite symmetric, lacking the characteristic 350-nm shoulder found in other rubredoxins. The flavodoxin is a 14,500-molecular-weight protein which contains 1 mol of flavin mononucleotide per mol of protein. It forms a stable, blue semiquinone upon light irradiation in the presence of EDTA or during enzymatic reduction. When cells were grown in low-iron medium, flavodoxin constituted at least 2% of the soluble cell protein; however, it was not detected in extracts of cells grown in high-iron medium. The rubredoxin and ferredoxin expressed during growth in low-iron and high-iron media are identical as judged by iron, inorganic sulfide, and amino acid analysis, as well as light absorption spectroscopy.

Isolation and characterization of two rubredoxins from Clostridium thermoaceticum.

Two rubredoxins with similar molecular weights (about 6000) have been purified from Clostridium thermoaceticum, a thermophile and strict anaerobe. They exhibit minor differences in several properties like elution pattern from DEAE-cellulose column, isoelectric point, amino acid composition, absorption and EPR spectra and redox potential. Their chemical and physical properties are similar to those of other rubredoxins from anaerobic microorganisms.

Amino acids and peptides. XLII. Synthesis of an octapeptide sequence (A20-A27) of rubredoxin.

A protected octapeptide corresponding to a section found in the first half of the protein chain of rubredoxin has been prepared by standard peptide methods. Alternative approaches of this fragment are discussed in some detail. This work completes the series of subunits necessary to synthesize the molecule.