Biomaterial Database

Mutated forms of the [2Fe-2S] ferredoxin from Clostridium pasteurianum with noncysteinyl ligands to the iron-sulfur cluster. 1994

J Meyer, and J Fujinaga, and J Gaillard, and M Lutz

CEA, Département de Biologie Moléculaire et Structurale, CENG, Grenoble, France.

The [2Fe-2S] ferredoxin from Clostridium pasteurianum is unique among ferredoxins, both by its sequence and by the distribution of its cysteine residues (in positions 11, 14, 24, 56, 60). Thus, no homologous sequences are available to infer, by comparison, the identity of the ligands of the iron-sulfur cluster. Therefore, in order to obtain information on the latter point, a combination of site-directed mutagenesis and UV-vis, EPR, and resonance Raman spectroscopy has been implemented. All of the cysteine residues have individually been replaced by serine and two of them by alanine. Cysteine 14 could be replaced by either serine or alanine without any modification of the spectroscopic properties of the protein and was therefore dismissed as a ligand of the [2Fe-2S] cluster. The C56S, and C60S-mutated proteins were both found to display UV-vis, EPR, and resonance Raman spectra consistent with serine-coordinated [2Fe-2S] clusters. The C11S-mutated protein was considerably less stable than the wild type ferredoxin. This observation, together with the hypsochromic shifts of UV-visible absorption features upon cysteine 11-->serine mutation, suggested cysteine 11 to be a ligand of the [2Fe-2S] cluster. Cysteine 24 could be replaced by either serine or alanine without decreasing the stability of the protein and without dramatically changing its spectroscopic properties. Thus, either cysteine 24 is not a ligand of the [2Fe-2S] cluster or it is replaced by another ligand in the C24A mutated protein. A [2Fe-2S] cluster was also assembled in the C14A/C24A doubly mutated protein, i.e., in a polypeptide chain containing only three cysteine residues.2+ off

UI	MeSH Term	Description	Entries
D007501	Iron	A metallic element with atomic symbol Fe, atomic number 26, and atomic weight 55.85. It is an essential constituent of HEMOGLOBINS; CYTOCHROMES; and IRON-BINDING PROTEINS. It plays a role in cellular redox reactions and in the transport of OXYGEN.	Iron-56,Iron 56
D008024	Ligands	A molecule that binds to another molecule, used especially to refer to a small molecule that binds specifically to a larger molecule, e.g., an antigen binding to an antibody, a hormone or neurotransmitter binding to a receptor, or a substrate or allosteric effector binding to an enzyme. Ligands are also molecules that donate or accept a pair of electrons to form a coordinate covalent bond with the central metal atom of a coordination complex. (From Dorland, 27th ed)	Ligand
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D003013	Clostridium	A genus of motile or nonmotile gram-positive bacteria of the family Clostridiaceae. Many species have been identified with some being pathogenic. They occur in water, soil, and in the intestinal tract of humans and lower animals.
D003545	Cysteine	A thiol-containing non-essential amino acid that is oxidized to form CYSTINE.	Cysteine Hydrochloride,Half-Cystine,L-Cysteine,Zinc Cysteinate,Half Cystine,L Cysteine
D004269	DNA, Bacterial	Deoxyribonucleic acid that makes up the genetic material of bacteria.	Bacterial DNA
D004578	Electron Spin Resonance Spectroscopy	A technique applicable to the wide variety of substances which exhibit paramagnetism because of the magnetic moments of unpaired electrons. The spectra are useful for detection and identification, for determination of electron structure, for study of interactions between molecules, and for measurement of nuclear spins and moments. (From McGraw-Hill Encyclopedia of Science and Technology, 7th edition) Electron nuclear double resonance (ENDOR) spectroscopy is a variant of the technique which can give enhanced resolution. Electron spin resonance analysis can now be used in vivo, including imaging applications such as MAGNETIC RESONANCE IMAGING.	ENDOR,Electron Nuclear Double Resonance,Electron Paramagnetic Resonance,Paramagnetic Resonance,Electron Spin Resonance,Paramagnetic Resonance, Electron,Resonance, Electron Paramagnetic,Resonance, Electron Spin,Resonance, Paramagnetic
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005288	Ferredoxins	Iron-containing proteins that transfer electrons, usually at a low potential, to flavoproteins; the iron is not present as in heme. (McGraw-Hill Dictionary of Scientific and Technical Terms, 5th ed)	Ferredoxin,Ferredoxin I,Ferredoxin II,Ferredoxin III