scholarly journals Structural studies on Desulfovibrio gigas cytochrome c3 by two-dimensional 1H-nuclear-magnetic-resonance spectroscopy

1993 ◽  
Vol 294 (3) ◽  
pp. 909-915 ◽  
Author(s):  
M A Piçarra-Pereira ◽  
D L Turner ◽  
J LeGall ◽  
A V Xavier
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Reduced Form ◽  
Desulfovibrio Vulgaris ◽  
Resonance Spectroscopy ◽  
Two Dimensional ◽  
Redox Potentials ◽  
Cytochrome C3 ◽  
X Ray ◽  
Desulfovibrio Gigas

Several aromatic amino acid residues and haem resonances in the fully reduced form of Desulfovibrio gigas cytochrome c3 are assigned, using two-dimensional 1H n.m.r., on the basis of the interactions between the protons of the aromatic amino acids and the haem protons as well as the intrahaem distances known from the X-ray structure [Kissinger (1989) Ph.D. Thesis, Washington State University]. The interhaem interactions observed in the n.m.r. spectra are in full agreement with the D. gigas X-ray structure and also with the n.m.r. data from Desulfovibrio vulgaris (Hildenborough) [Turner, Salgueiro, LeGall and Xavier (1992) Eur. J. Biochem. 210, 931-936]. The good correlation between the calculated ring-current shifts and the observed chemical shifts strongly supports the present assignments. Observation of the two-dimensional nuclear-Overhauser-enhancement spectra of the protein in the reduced, intermediate and fully oxidized stages led to the ordering of the haems in terms of their midpoint redox potentials and their identification in the X-ray structure. The first haem to oxidize is haem I, followed by haems II, III and IV, numbered according to the Cys ligand positions in the amino acid sequences [Mathews (1985) Prog. Biophys. Mol. Biol. 54, 1-56]. Although the haem core architecture is the same for the different Desulfovibrio cytochromes c3, the order of redox potentials is different.

Resolution of a protein sequence ambiguity by X-ray crystallographic and mass spectrometric methods

1992 ◽  
Vol 25 (2) ◽  
pp. 205-210 ◽  
Author(s):  
L. J. Keefe ◽  
E. E. Lattman ◽  
C. Wolkow ◽  
A. Woods ◽  
M. Chevrier ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Electron Density ◽  
Mass Spectrometric ◽  
Amino Acid Sequences ◽  
Data Matrix ◽  
Protein Crystal ◽  
Insertion Mutant ◽  
Laser Desorption Mass Spectrometry ◽  
X Ray

Ambiguities in amino acid sequences are a potential problem in X-ray crystallographic studies of proteins. Amino acid side chains often cannot be reliably identified from the electron density. Many protein crystal structures that are now being solved are simple variants of a known wild-type structure. Thus, cloning artifacts or other untoward events can readily lead to cases in which the proposed sequence is not correct. An example is presented showing that mass spectrometry provides an excellent tool for analyzing suspected errors. The X-ray crystal structure of an insertion mutant of Staphylococcal nuclease has been solved to 1.67 Å resolution and refined to a crystallographic R value of 0.170 [Keefe & Lattman (1992). In preparation]. A single residue has been inserted in the C-terminal α helix. The inserted amino acid was believed to be an alanine residue, but the final electron density maps strongly indicated that a glycine had been inserted instead. To confirm the observations from the X-ray data, matrix-assisted laser desorption mass spectrometry was employed to verify the glycine insertion. This mass spectrometric technique has sufficient mass accuracy to detect the methyl group that distinguishes glycine from alanine and can be extended to the more common situation in which crystallographic measurements suggest a problem with the sequence, but cannot pinpoint its location or nature.

Elucidation of the two-dimensional structure of an α-amino acid surfactant monolayer on water using synchrotron X-ray diffraction

Nature ◽  
1987 ◽  
Vol 328 (6125) ◽  
pp. 63-66 ◽  
Author(s):  
Sharon Grayer Wolf ◽  
Leslie Leiserowitz ◽  
Meir Lahav ◽  
Moshe Deutsch ◽  
Kristian Kjaer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dimensional Structure ◽  
Two Dimensional ◽  
X Ray Diffraction ◽  
X Ray

scholarly journals Characterization of the structure and redox behaviour of cytochrome c3 from Desulfovibrio baculatus by 1H-nuclear-magnetic-resonance spectroscopy

1993 ◽  
Vol 294 (3) ◽  
pp. 899-908 ◽  
Author(s):  
I B Coutinho ◽  
D L Turner ◽  
J LeGall ◽  
A V Xavier
Keyword(s):  
Chemical Shifts ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Resonance Spectroscopy ◽  
Cytochrome C3 ◽  
X Ray ◽  
1H Nuclear Magnetic Resonance ◽  
Proton Resonances ◽  
Nuclear Overhauser

Complete assignment of the aromatic and haem proton resonances in the cytochromes c3 isolated from Desulfovibrio baculatus strains (Norway 4, DSM 1741) and (DSM 1743) was achieved using one- and two-dimensional 1H n.m.r. Nuclear Overhauser enhancements observed between haem and aromatic resonances and between resonances due to different haems, together with the ring-current contributions to the chemical shifts of haem resonances, support the argument that the haem core architecture is conserved in the various cytochromes c3, and that the X-ray structure of the D. baculatus cytochrome c3 is erroneous. The relative orientation of the haems for both cytochromes was determined directly from n.m.r. data. The n.m.r. structures have a resolution of approximately 0.25 nm and are found to be in close agreement with the X-ray structure from D. vulgaris cytochrome c3. The proton assignments were used to relate the highest potential to a specific haem in the three-dimensional structure by monitoring the chemical-shift variation of several haem resonances throughout redox titrations followed by 1H n.m.r. The haem with highest redox potential is not the same as that in other cytochromes c3.

scholarly journals Characterization of Hydrogenase II from the Hyperthermophilic Archaeon Pyrococcus furiosus and Assessment of Its Role in Sulfur Reduction

2000 ◽  
Vol 182 (7) ◽  
pp. 1864-1871 ◽  
Author(s):  
Kesen Ma ◽  
Robert Weiss ◽  
Michael W. W. Adams
Keyword(s):  
Amino Acid ◽  
Elemental Sulfur ◽  
Pyrococcus Furiosus ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Amino Acid Sequences ◽  
Electron Donors ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Nucleotide Binding Sites ◽  
Γ Subunit

ABSTRACT The fermentative hyperthermophile Pyrococcus furiosuscontains an NADPH-utilizing, heterotetrameric (αβγδ), cytoplasmic hydrogenase (hydrogenase I) that catalyzes both H2 production and the reduction of elemental sulfur to H2S. Herein is described the purification of a second enzyme of this type, hydrogenase II, from the same organism. Hydrogenase II has an M r of 320,000 ± 20,000 and contains four different subunits withM rs of 52,000 (α), 39,000 (β), 30,000 (γ), and 24,000 (δ). The heterotetramer contained Ni (0.9 ± 0.1 atom/mol), Fe (21 ± 1.6 atoms/mol), and flavin adenine dinucleotide (FAD) (0.83 ± 0.1 mol/mol). NADPH and NADH were equally efficient as electron donors for H2 production withKm values near 70 μM andk cat/Km values near 350 min−1 mM−1. In contrast to hydrogenase I, hydrogenase II catalyzed the H2-dependent reduction of NAD (Km , 128 μM;k cat/Km , 770 min−1 mM−1). Ferredoxin from P. furiosus was not an efficient electron carrier for either enzyme. Both H2 and NADPH served as electron donors for the reduction of elemental sulfur (S0) and polysulfide by hydrogenase I and hydrogenase II, and both enzymes preferentially reduce polysulfide to sulfide rather than protons to H2using NADPH as the electron donor. At least two [4Fe-4S] and one [2Fe-2S] cluster were detected in hydrogenase II by electron paramagnetic resonance spectroscopy, but amino acid sequence analyses indicated a total of five [4Fe-4S] clusters (two in the β subunit and three in the δ subunit) and one [2Fe-2S] cluster (in the γ subunit), as well as two putative nucleotide-binding sites in the γ subunit which are thought to bind FAD and NAD(P)(H). The amino acid sequences of the four subunits of hydrogenase II showed between 55 and 63% similarity to those of hydrogenase I. The two enzymes are present in the cytoplasm at approximately the same concentration. Hydrogenase II may become physiologically relevant at low S0concentrations since it has a higher affinity than hydrogenase I for both S0 and polysulfide.

scholarly journals Electron transfer to nitrogenase. Characterization of flavodoxin from Azotobacter chroococcum and comparison of its redox potentials with those of flavodoxins from Azotobacter vinelandii and Klebsiella pneumoniae (nifF-gene product)

1986 ◽  
Vol 239 (1) ◽  
pp. 69-75 ◽  
Author(s):  
J Deistung ◽  
R N F Thorneley
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Gene Product ◽  
Azotobacter Vinelandii ◽  
Azotobacter Chroococcum ◽  
Amino Acid Sequences ◽  
Published Data ◽  
Redox Potentials ◽  
Hydrogen Electrode ◽  
Visible Spectra

Flavodoxin in the hydroquinone state acts as an electron donor to nitrogenase in several nitrogen-fixing organisms. The mid-point potentials for the oxidized-semiquinone and semiquinone-hydroquinone couples of flavodoxins isolated from facultative anaerobe Klebsiella pneumoniae (nifF-gene product, KpFld) and the obligate aerobe Azotobacter chroococcum (AcFld) were determined as a function of pH. The mid-point potentials of the semiquinone-hydroquinone couples of KpFld and AcFld are essentially independent of pH over the range pH 7-9, being -422 mV and -522 mV (normal hydrogen electrode) at pH 7.5 respectively. The mid-point potentials of the quinone-semiquinone couples at pH 7.5 are -200 mV (KpFld) and -133 mV (AcFld) with delta Em/pH of -65 +/- 4 mV (KpFld) and -55 +/- 2 mV (AcFld) over the range pH 7.0-9.5. This indicates that reduction of the quinone is coupled to protonation to yield a neutral semiquinone. The significance of these values with respect to electron transport to nitrogenase is discussed. The amino acid compositions, the N- and C-terminal amino acid sequences and the u.v.-visible spectra of KpFld and AcFld were determined and are compared with published data for flavodoxins isolated from Azotobacter vinelandii.

Synopses from the poster exhibition organized by I. M. Kerr, 1. Interferon: a tertiary structure predicted from amino acid sequences

1982 ◽  
Vol 299 (1094) ◽  
pp. 125-127 ◽  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Tertiary Structure ◽  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
X Ray ◽  
Three Dimensional Structure ◽  
X Ray Crystallography ◽  
Functional Studies

Functional studies on interferon would be helped by a three-dimensional structure for the molecule. However, it may be several years before the structure of the protein is determined by X-ray crystallography. We have therefore used available methods for predicting the secondary - and the tertiary - structure of a protein from its amino acid sequence to propose a tertiary model involving the packing of four a-helices. Details of this work have been published elsewhere (Sternberg & Cohen 1982).

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