The purification, amino acid sequence, and two-dimensional 1H NMR results are reported for the rubredoxin (Rd) from the hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 degrees C. The molecular mass (5397 Da), iron content (1.2 +/- 0.2 g-atom of Fe/mol), UV-vis spectrophotometric properties, and amino acid sequence (60% sequence identity with Clostridium pasteurianum Rd) are found to be typical of this class of redox protein. However, P. furiosus Rd is remarkably thermostable, being unaffected after incubation for 24 h at 95 degrees C. One- and two-dimensional 1H nuclear magnetic resonance spectra of the oxidized [Fe(III)Rd] and reduced [Fe(II)Rd] forms of P. furiosus Rd exhibited substantial paramagnetic line broadening, and this precluded detailed 3D structural studies. The apoprotein was not readily amenable to NMR studies due to apparent protein oxidation involving the free cysteine sulfhydryls. However, high-quality NMR spectra were obtained for the Zn-substituted protein, Zn(Rd), enabling detailed NMR signal assignment for all backbone amide and alpha and most side-chain protons. Secondary structural elements were determined from qualitative analysis of 2D Overhauser effect spectra. Residues A1-K6, Y10-E14, and F48-E51 form a three-strand antiparallel beta-sheet, which comprises ca. 30% of the primary sequence. Residues C5-Y10 and C38-A43 form types I and II amide-sulfur tight turns common to iron-sulfur proteins. These structural elements are similar to those observed by X-ray crystallography for native Rd from the mesophile C. pasteurianum. However, the beta-sheet domain in P. furiosus Rd is larger than that in C. pasteurianum Rd and appears to begin at the N-terminal residue. From analysis of the secondary structure, potentially stabilizing electrostatic interactions involving the charged groups of residues Ala(1), Glu(14), and Glu(52) are proposed. These interactions, which are not present in rubredoxins from mesophilic organisms, may prevent the beta-sheet from "unzipping" at elevated temperatures.

Ciliary and flagellar axonemes are basically composed of nine outer doublet microtubules and several functional components, e.g. dynein arms, radial spokes, and interdoublet links. Each A-tubule of the doublet contains a specialized "ribbon" of three protofilaments composed of tubulin and other proteins postulated to specify the three-dimensional arrangement of the various axonemal components. The interdoublet links hold the doublet microtubules together and limit their sliding during the flagellar beat. In this study on Chlamydomonas reinhardtii, we cloned a cDNA encoding a 71,985-Da polypeptide with three DM10 repeats, two C-terminal EF-hand motifs, and homologs extending to humans. This polypeptide, designated as Rib72, is a novel component of the ribbon compartment of flagellar microtubules. It remained associated with 9-fold arrays of doublet tubules following extraction under high and low ionic conditions, and anti-Rib72 antibodies revealed an ϳ96-nm periodicity along axonemes, consistent with Rib72 associating with interdoublet links. Following proteolysis-and ATP-dependent disintegration of axonemes, the rate of cleavage of Rib72 correlated closely with the rate of sliding disintegration. These observations identify a ribbon-associated protein that may function in the structural assembly of the axoneme and in the mechanism and regulation of ciliary and flagellar motility.

The iron and acid-labile sulfide contents and the electron paramagnetic resonance (EPR) properties of hydrogenase I (bidirectional) and hydrogenase II (uptake) of Clostridiumpasteurianum (strain W5) have been determined on the basis of quantitative amino acid analyses. The iron and acid-labile sulfide values are approximately 20 and 18 atoms per molecule of hydrogenase I and 14 and 11 atoms per molecule of hydrogenase II, respectively. These amounts are substantially greater than previously reported values, which relied on protein concentration determined by calorimetric assay. The oxidized hydrogenases exhibit unusual EPR signals that originate from a novel type of iron-sulfur center, termed the hydrogenase or H cluster, which covalently binds the inhibitor CO. This EPR signal represents approximately one unpaired electron per molecule in each enzyme with and without bound CO, which is consistent with the presence of one oxidized H cluster (S = 1/2) per enzyme molecule. The two enzymes also contain ferredoxin-type four-iron centers or F clusters. The EPR signals from the F clusters observed in the reduced forms ofhydrogenase I and hydrogenase II account for approximately four and one unpaired electron per molecule, respectively. We conclude from the iron determinations and the EPR results, together with a reevaluation of previous spectroscopic data, that in both hydrogenases the H cluster probably comprises six iron atoms. Mechanistic models of the two hydrogenases are presented that account for their cluster compositions and the dramatic differences in their catalytic activities.Hydrogenases catalyze the reversible activation of molecular H2 according to the reaction:They are widely distributed in bacteria and are a very heterogeneous group of enzymes: their only common feature is that they are iron-sulfur proteins and the majority also contain nickel (1-4). The anaerobic N2-fixing bacterium Clostridium pasteurianum is so far unique in that it contains two hydrogenases that lack nickel. Hydrogenase I (bidirectional) catalyzes both H2 evolution and H2 oxidation at extremely high rates in vitro, whereas hydrogenase II preferentially catalyzes H2 oxidation (5)(6)(7)(8) (7,(9)(10)(11)(12). A combination of EPR and 1H, 13C, and 57Fe electron nuclear double resonance spectroscopy (ENDOR) has shown that in both hydrogenases the H cluster contains two types of iron atoms and covalently binds CO, a potent inhibitor, without cluster destruction (13,14). Subsequent Mossbauer analysis of hydrogenase II showed that its H cluster contains two types of iron atoms in a 2:1 ratio and was '40% of the total iron (15). Therefore, it was concluded that the H cluster is a three-iron cluster (15). Thus, on the basis of these results and the determined iron concentrations, hydrogenase II was thought to contain 7 iron atoms per molecule (one H and one F cluster), and hydrogenase I, 11 iron atoms per molecule (one H and two F clusters). However, the proposed cluster compositions of the clostridial hydrogenases are not consisten...

The principal galactose oxidase/NaB[3H]4-labeled membrane protein of rat caudal epididymal spermatozoa was isolated by hydrophobic interaction chromatography. The protein is released from the membrane by the action of phosphatidylinositol specific phospholipase C, and thereby its properties are transformed from those of a protein anchored to the hydrophobic membrane to those of a hydrophilic solution protein. Because it is the only membrane-associated protein released by the enzyme which did not absorb to a propylaspartate resin, a simple, single step purification procedure was devised. Although the amino terminus of the protein is blocked to Edman degradation, the majority of the protein structure was determined from a series of tryptic peptides and from limited acid hydrolysis. Approximately 65% of the protein mass is carbohydrate which is primarily attached through O-glycosidic bonds to the 18 threonines. The molecular weight of the glycoprotein was estimated to be 16,600, considerably smaller than the M(r) = 26,000 to 37,000 previously determined by gel electrophoresis. The anomalous electrophoretic behavior is undoubtedly due to the large percentage of carbohydrate. The distribution of carbohydrate on the protein side chains suggests the protein may form a positively charged, specialized scaffolding for the presentation of the carbohydrate moieties. Because the appearance of the ability to label the protein with galactose oxidase is correlated with sperm maturation in the epididymis, the glycoprotein structures may be an important component in the fertilization process. The combination of linkage by glycosylphosphatidylinositol and low molecular weight mucin-like structure indicates this may be a member of a new class of membrane proteins.

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