The Rubredoxin from Clostridium pasteurianum: Mutation of the Conserved Glycine Residues 10 and 43 to Alanine and Valine
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- 作者：Mustafa Ayhan ; Zhiguang Xiao ; Megan J. Lavery ; Amanda M. Hamer ; Kerry W. Nugent ; Sergio D. B. Scrofani ; Mitchell Guss ; and Anthony G. Wedd
- 刊名：Inorganic Chemistry
- 出版年：1996
- 出版时间：September 25, 1996
- 年：1996
- 卷：35
- 期：20
- 页码：5902 - 5911
- 全文大小：333K
- 年卷期：v.35,no.20(September 25, 1996)
- ISSN：1520-510X
文摘
Conserved glycine residues at positions 10 and 43 in theelectron transfer protein rubredoxin (active site:Fe(Cys-S)4) from Clostridiumpasteurianum are related by a pseudo-2-fold symmetry. Theyhave been mutated toalanine and valine and four single and two double mutant (G10V/G43A andG10V/G43V) proteins expressed instable form in Escherichia coli. Physical propertieswere modified by steric interactions between the 
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-carbon substituents of the new side chains and the CO functions ofC9 and C42 and other adjacent groups.These interactions perturb the chelate loops formed by residues5-11 and 38-44. 1H NMR results forCd(II)forms indicate that the Pri side chain of V10 in the G10Vmutant occupies the surface pocket defined by loop5-11 and thereby modifies the environment of the 5-11 NH protons.The equivalent side chain of V43 inG43V is denied the same access to the 38-44 pocket. This leadsto a specific perturbation of the V44-NH···S-C42 interaction in this mutant. These effects are additive in thedouble mutant G10V/G43V, consistent with thedifferent structural changes being localized in each loop. Themidpoint potentials of the iron forms of the sixmutants are shifted negatively relative to the recombinant protein by-16 to -86 mV. A G 
V mutation hasa larger effect than a G A, but again, an additivity of thedifferential effects is seen in the double mutants.Minor perturbations of resonance Raman and electronic spectra aredominated by the mutation at G10. Overall,the present work represents one approach to the systematic explorationof the influence of the protein chain uponthe fundamental properties of this molecule.
- and-carbon substituents of the new side chains and the CO functions ofC9 and C42 and other adjacent groups.These interactions perturb the chelate loops formed by residues5-11 and 38-44. 1H NMR results forCd(II)forms indicate that the Pri side chain of V10 in the G10Vmutant occupies the surface pocket defined by loop5-11 and thereby modifies the environment of the 5-11 NH protons.The equivalent side chain of V43 inG43V is denied the same access to the 38-44 pocket. This leadsto a specific perturbation of the V44-NH···S-C42 interaction in this mutant. These effects are additive in thedouble mutant G10V/G43V, consistent with thedifferent structural changes being localized in each loop. Themidpoint potentials of the iron forms of the sixmutants are shifted negatively relative to the recombinant protein by-16 to -86 mV. A G V mutation hasa larger effect than a G A, but again, an additivity of thedifferential effects is seen in the double mutants.Minor perturbations of resonance Raman and electronic spectra aredominated by the mutation at G10. Overall,the present work represents one approach to the systematic explorationof the influence of the protein chain uponthe fundamental properties of this molecule.