Zinc- and iron-rubredoxins from Clostridium pasteurianum at atomic resolution: a high-precision model of a ZnS4 coordination unit in a protein.

Dauter, Zbigniew; Wilson, K.S.; Sieker, L.C.; Moulis, Jean‐Marc; Meyer, Jacques

doi:10.1073/pnas.93.17.8836

Cited by 148 publications

(197 citation statements)

References 38 publications

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“…Similar observations have been reported for recombinant Rds heterologously overproduced in E. coli in which zinc occupies the iron site (32)(33)(34)(35). 2 We used the ironenriched triple variant (prepared by supplying 150 -200 M FeCl 3 in the growth medium during overexpression) in the following analyses.…”

Section: Resultsmentioning

confidence: 76%

Rational Design of a Mononuclear Metal Site into the Archaeal Rieske-type Protein Scaffold

Iwasaki

Kounosu

et al. 2005

Journal of Biological Chemistry

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Proteins containing Rieske-type [2Fe-2S] clusters play essential functions in all three domains of life. We engineered the two histidine ligands to the Rieske-type [2Fe-2S] cluster in the hyperthermophilic archaeal Riesketype ferredoxin from Sulfolobus solfataricus to modify types and spacing of ligands and successfully converted the metal and cluster type at the redox-active site with a minimal structural change to a native Rieske-type protein scaffold. Spectroscopic analyses unambiguously established a rubredoxin-type mononuclear Fe 3؉/2؉ center at the engineered local metal-binding site (Zn 2؉ occupies the iron site depending on the expression conditions). These results show the importance of types and spacing of ligands in the in vivo cluster recognition/insertion/assembly in biological metallosulfur protein scaffolds. We suggest that early ligand substitution and displacement events at the local metal-binding site(s) might have primarily allowed the metal and cluster type conversion in ancestral redox protein modules, which greatly enhanced their capabilities of conducting a wide range of unique redox chemistry in biological electron transfer conduits, using a limited number of basic protein scaffolds.Natural selection allows the utilization of a limited number of protein scaffolds to produce proteins with different types of active sites for various biological catalysis, molecular recognition, and metabolic needs. Metal ions add new functionality to proteins, facilitating some of the most difficult biological catalytic reactions (1-3). For this reason, design and engineering of a specific metalbinding site in natural and de novo protein scaffolds to promote new and specific functionalities are attractive targets in the field of basic and applied protein design research (4, 5).The metallosulfur redox sites, containing sulfurs, usually from cysteinyl side chains, are the most common in biological redox-active metalloproteins (1). In particular, the iron-sulfur (Fe-S) proteins are widely distributed over all living organisms and have been considered to be of early evolutionary origin (2, 6, 7). The mononuclear iron core is the simplest form of Fe-S redox sites, present in small modular proteins such as rubredoxin (Rd) 1 and desulforedoxin. These proteins have the iron atom coordinated in an approximately tetrahedral geometry to the sulfur atoms of four cysteinyl residues (1). Other major forms of protein-bound Fe-S redox sites are polynuclear clusters (such as [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters) for which some specific synthesis/assembly enzymes are required for in vivo cluster formation and maturation steps (7-10). These sites are also found within complex metalloprotein molecules themselves where they form part of the internal electron transfer conduit to or from the catalytic site (e.g. in some membrane-bound respiratory complexes (11-13)) as a result of modular evolution.Among the biological Fe-S clusters with at least one noncysteinyl ligand, "Rieske-type" [2Fe-2S] clusters are ubiquitous in a vari...

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Section: Resultsmentioning

confidence: 76%

Rational Design of a Mononuclear Metal Site into the Archaeal Rieske-type Protein Scaffold

Iwasaki

Kounosu

et al. 2005

Journal of Biological Chemistry

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“…Residues relevant to this study are indicated. The drawing was generated using Rasmol, from coordinates in file 1IRO in the Protein Data Bank [34] Bonomi et al intensities at 495 nm, and scaling to the corresponding spectral intensities of the appropriate holoRd Electrospray ionization mass spectrometry (ESI-MS) spectra of apoRds, holoRds or a mixture containing apoRd, 6 M chaotrope and stochiometric iron. Right: CpRd (6 M urea as the chaotrope).…”

Section: Supplementary Materialsmentioning

confidence: 99%

“Iron priming” guides folding of denatured aporubredoxins

Bonomi¹,

Iametti²,

Ferranti³

et al. 2008

J Biol Inorg Chem

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The relationship between iron uptake by aporubredoxins (apoRds) and formation of native holorubredoxins (holoRd), including their Fe(SCys) 4 sites, was studied. In the absence of denaturants, apoRds exhibited spectroscopic features consistent with structures very similar to those of the folded holoRds. However, additions of either ferric or ferrous salts to the apoRds in the absence of denaturants gave less than 40% recovery of the native holoRd circular dichroism and UV-vis spectroscopic features. In the presence of either 6 M urea or 6 M guanidine hydrochloride, the nativelike structural features of the apoRds were absent. Nevertheless, nearly quantitative recoveries of the native holoRd spectroscopic features were achieved by addition of either ferric or ferrous salts to the denatured apoRds without diluting the denaturant. Consistent with this observation, the native spectroscopic features were unaffected by addition of the same denaturant concentrations to the as-isolated holoRds. Denaturing concentrations of urea or guanidine hydrochloride also increased the rates of holoRd recoveries from apoRds and ferrous salts. Mass spectrometry confirmed that ferric iron binding to the denatured apoRds precedes the recoveries of protein secondary structures and Fe(SCys) 4 sites. Thus, iron binding to the apoRds guides, both kinetically and thermodynamically, refolding to the native holoRd structures. Our results imply that the ferrous oxidation state would more efficiently drive formation of the native holoRd structure from the nascent apoprotein in vivo, but that the Fe(SCys) 4 site must attain the ferric state in order to achieve its native structure.Correspondence to: Francesco Bonomi. Electronic supplementary material The online version of this article

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“…The refinement of one of these, from Desulfovibrio vulgaris, has been extended to a 1.0-Å resolution (38), and that data set was used in a study describing the application of direct methods to protein crystallography (39). All of those studies indicated that rubredoxins contain a very small three-stranded antiparallel ␤-sheet, a hydrophobic core, and several loops (29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39).…”

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confidence: 99%

“…That analysis, carried out at a resolution of 1.5 Å, featured the first report of a least-squares refinement of a protein at a truly atomic level. The structural analysis of rubredoxin from C. pasteurianum was eventually extended to 1.2-Å resolution (31) and 1.1-Å resolution (32), and other rubredoxin structures have been reported over the years from other mesophilic (room temperature) organisms (33)(34)(35)(36)(37)(38)(39)). The refinement of one of these, from Desulfovibrio vulgaris, has been extended to a 1.0-Å resolution (38), and that data set was used in a study describing the application of direct methods to protein crystallography (39).…”

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confidence: 99%

Neutron crystallographic study on rubredoxin from Pyrococcus furiosus by BIX-3, a single-crystal diffractometer for biomacromolecules

Kurihara

Tanaka

Chatake

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Zinc- and iron-rubredoxins from Clostridium pasteurianum at atomic resolution: a high-precision model of a ZnS4 coordination unit in a protein.

Cited by 148 publications

References 38 publications

Rational Design of a Mononuclear Metal Site into the Archaeal Rieske-type Protein Scaffold

Rational Design of a Mononuclear Metal Site into the Archaeal Rieske-type Protein Scaffold

“Iron priming” guides folding of denatured aporubredoxins

Neutron crystallographic study on rubredoxin from Pyrococcus furiosus by BIX-3, a single-crystal diffractometer for biomacromolecules

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