Crystallographic studies of V44 mutants of <i>Clostridium pasteurianum</i> rubredoxin: Effects of side‐chain size on reduction potential

Park, Il Yeong; Eidsness, Marly K.; Lin, I-Jin; Gebel, Erika; Youn, BuHyun; Harley, J.L.; Machonkin, Timothy E.; Frederick, Ronnie O.; Markley, John L.; Smith, Eugene T.; Ichiye, Toshiko; Kang, ChulHee

doi:10.1002/prot.20243

Cited by 21 publications

(32 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Detailed comparison of available x-ray crystal structures of CpRd variants has revealed that slight changes in the structure surrounding the iron-sulfur cluster are responsible for the range of observed reduction potentials (15,16,28). In our study, 15 N hyperfine shifts were used to detect subtle changes in H N ⅐⅐⅐⅐⅐S ␥ bond lengths in CpRd variants.…”

Section: Discussionmentioning

confidence: 89%

Changes in hydrogen-bond strengths explain reduction potentials in 10 rubredoxin variants

Lin¹,

Gebel²,

Machonkin³

et al. 2005

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

Self Cite

113

View full text Add to dashboard Cite

The rubredoxin from Clostridium pasteurianum (CpRd) provides an excellent system for investigating how the protein sequence modulates the reduction potential of the active site in an iron-sulfur protein. 15 N NMR spectroscopy has allowed us to determine with unprecedented accuracy the strengths of all six key hydrogen bonds between protein backbone amides and the sulfur atoms of the four cysteine residues that ligate the iron in the oxidized (Fe III ) and reduced (Fe II ) forms of wild-type CpRd and nine mutants (V44G, V44A, V44I, V44L, V8G, V8A, V8I, V8L, and V8G͞V44G). The length (or strength) of each hydrogen bond was inferred from the magnitude of electron spin delocalized across the hydrogen bond from the iron atom onto the nitrogen. The aggregate lengths of these six hydrogen bonds are shorter in both oxidation states in variants with higher reduction potential than in those with lower reduction potential. Differences in aggregate hydrogen bonding upon reduction correlate linearly with the published reduction potentials for the 10 CpRd variants, which span 126 mV. Sequence effects on the reduction potential can be explained fully by their influence on hydrogen-bond strengths.iron-sulfur protein ͉ reduction potential tuning ͉ 15 N NMR ͉ paramagnetic NMR

show abstract

Section: Discussionmentioning

confidence: 89%

Changes in hydrogen-bond strengths explain reduction potentials in 10 rubredoxin variants

Lin¹,

Gebel²,

Machonkin³

et al. 2005

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

Self Cite

113

View full text Add to dashboard Cite

show abstract

“…16,[29][30][31][32][33] We distinguish three types of H-bonds ( Figure 1a) (HB1, HB2, HB3, depicted in Scheme 1). Each of the ISC sulfur atoms, S1 and S3, is engaged in two H-bonds, which we call type HB1 and HB2.…”

Section: Isc H-bonds In Rdmentioning

confidence: 99%

Understanding Rubredoxin Redox Potentials: Role of H-Bonds on Model Complexes

Gámiz‐Hernández

Galstyan

Knapp

2009

J. Chem. Theory Comput.

View full text Add to dashboard Cite

The energetics of redox states in different models of rubredoxin-like iron-sulfur complexes (ISC) was computed using a combination of density functional and electrostatic continuum theories. In agreement with experiment, the calculated redox potential for the small ISC model [Fe(SCH2CH3)4](1-,2-) in acetonitrile was -813 mV [Galstyan, A. S.; Knapp, E. W. J. Comput. Chem. 2009, 30, 203-211] as compared to the measured value of -838 mV. Surprisingly the experimental values for rubredoxin (Rd) are much higher ranging between -87 and +39 mV. These large variations in redox potentials of ISC models and ISC in Rd are due to specific conformational symmetries adopted by the ligands due to both the protein environment and type and the number of H-bonds, and the dielectric environment. In a dielectric environment corresponding to proteins (ε = 20), the computed ISC redox potentials shift positive by about 64 mV for Fe-S···H-N and 95 mV for Fe-S···H-O H-bonds, correlating well with data estimated from experiments on ISC proteins. In aqueous solutions (ε = 80), a positive shift of 58 mV was computed for Fe-S···H-O H-bonds (using a model with the same ISC conformation as in Rd) in agreement with a measured value for Rd with partially solvent exposed ISC. The latter demonstrates the dependence of the ISC redox potentials on the environment (solvent or protein). For a model whose chemical composition is analog to the relevant part of ISC in a specific Rd, the computed redox potential of the model agrees with the measured value in Rd. This study allows to understand redox potential shifts for small ISC models and ISC in proteins.

show abstract

“…Investigations of thermally induced unfolding of CpRd showed that loss of the metal ion immediately precedes formation of the (apparently) unstructured apoRd [19], but that Fe(SCys) 4 site stability does not control the thermostability of the native folded structure [20]. Engineered V ↔ A exchanges of residue 44, which forms an N-H…S hydrogen bond to one of the ironcoordinating Cys residues, significantly raised and lowered the respective stabilities of the Fe(SCys) 4 sites in CpRd and PfRd [21][22][23][24] but had only minor effects on the thermal stabilities of the folded holoRds. Thiophilic metal ions (Cd II , Zn II ) can directly and isomorphically displace iron from the native Fe II (SCys) 4 site of CpRd under nondenaturing conditions and without unzipping of the β-sheet [25].…”

Section: Introductionmentioning

confidence: 99%

“Iron priming” guides folding of denatured aporubredoxins

Bonomi¹,

Iametti²,

Ferranti³

et al. 2008

J Biol Inorg Chem

View full text Add to dashboard Cite

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

show abstract

Crystallographic studies of V44 mutants of Clostridium pasteurianum rubredoxin: Effects of side‐chain size on reduction potential

Cited by 21 publications

References 23 publications

Changes in hydrogen-bond strengths explain reduction potentials in 10 rubredoxin variants

Changes in hydrogen-bond strengths explain reduction potentials in 10 rubredoxin variants

Understanding Rubredoxin Redox Potentials: Role of H-Bonds on Model Complexes

“Iron priming” guides folding of denatured aporubredoxins

Contact Info

Product

Resources

About