Expression of Pseudomonas stutzeri Zobell cytochrome c-551 and its H47A variant in Escherichia coli

Miller, Gregory T.; Mackay, Donald Q.; Standley, Melissa; Fields, Sherry L; Clary, Wendi M; Timkovich, Russell

doi:10.1016/s1046-5928(03)00065-2

Cited by 4 publications

(5 citation statements)

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“…The less efficient packing of residues around the heme propionates may account for the decreased acidic stability of H47A reported previously (3). His-47 in wild-type is positively charged under acidic conditions (2).…”

Section: Resultsmentioning

confidence: 80%

“…Fig. 7 (adopted from Milller et al (3)) displays this behavior for wild-type and the FIGURE 2 Schematic of the heme c prosthetic group with substituents numbered according to IUB-IUPAC nomenclature for tetrapyrroles. The additional small decimal numbers are the chemical shift differences, H47A minus wild-type, for heme protons.…”

Section: Resultsmentioning

confidence: 99%

“…The H47A mutant protein was expressed as described previously (3). The prior purification procedure yielded cytochrome c-551 free from other contaminating proteins, but it did contain peptidoglycan fragments released during the freeze-thaw steps that obscured important protein NMR resonances.…”

Section: Methodsmentioning

confidence: 99%

“…This conserved hydrogen bond may participate in heme redox potential modulation (2). The effect of residue 47 on protein stability and redox potential was studied by mutating His-47 to Ala-47 for Pseudomonas stutzeri substrain ZoBell cytochrome c-551 (3). The solution structure of H47A has now been determined by NMR and is reported herein.…”

Section: Introductionmentioning

confidence: 99%

“…FIGURE 7 Comparison of the pH dependence of the standard reduction potential for H47A (squares) and wild-type (diamonds) (data obtained fromMiller et al (3)). The smooth solid curves for each data set represent fitting the data with an equation whereby the pH dependence results from to a single ionization event that has different pK a values in the reduced and oxidized states as given by Leitch et al(19) with the parameters given by Miller et al(3). The dashed curve for H47A represents fitting the potential to simple Henderson-Hasselbalch behavior with a single pK a of 6.4 as described in the text and Supplemental Information.…”

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confidence: 99%

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Solution Conformation of the His-47 to Ala-47 Mutant of Pseudomonas stutzeri ZoBell Ferrocytochrome c-551

Liang

Miller

Beeghley

et al. 2007

Biophysical Journal

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In the cytochrome c-551 family, the heme 17-propionate caboxylate group is always hydrogen bonded to an invariant Trp-56 and conserved residues (His and Arg mainly, Lys occasionally) at position 47. The mutation of His-47 to Ala-47 for Pseudomas stutzeri ZoBell cytochrome c-551 removes this otherwise invariant hydrogen bond. The solution structure of ferrous H47A has been solved based on NMR-derived constraints. Results indicate that the mutant has very similar main chain folding compared to wild-type. However, less efficient packing of residues in the mutant surrounding the heme propionates leads to more solvent exposure for both propionate groups, which may account for decreased stability of the mutant. The mutant has a reduction potential different from wild-type, and furthermore, the pH dependence of this potential is not the same as for wild-type. The structure of the mutant suggests that these changes are related to the loss of the residue-47 propionate hydrogen bond and the loss of charge on the side chain of residue 47.

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Section: Resultsmentioning

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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confidence: 99%

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