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. 2006 Sep;89(2-3):89-98.
doi: 10.1007/s11120-006-9086-4. Epub 2006 Aug 17.

Redox properties of the Rhodobacter sphaeroides transcriptional regulatory proteins PpsR and AppA

S-K Kim  1 , J T Mason, D B Knaff, C E Bauer, A T Setterdahl
Affiliations

Redox properties of the Rhodobacter sphaeroides transcriptional regulatory proteins PpsR and AppA

S-K Kim et al. Photosynth Res. 2006 Sep.

Abstract

Redox properties of the photosynthetic gene repressor PpsR and the blue-light photoreceptor/antirepressor AppA from Rhodobacter sphaeroides have been characterized. Redox titrations of PpsR reveal the presence of a two-electron couple, with an E (m) value of -320 mV at pH 7.0, which is likely to arise from the reversible conversion of two cysteine thiols to a disulfide. This E (m) value is very much more negative than the E (m) = -180 mV value measured previously at pH 7.0 for the disulfide/dithiol couple in CrtJ, the homolog for PpsR in the closely related bacterium Rhodobacter capsulatus. AppA, a flavin-containing blue-light receptor that is also involved in the regulation of gene expression in R. sphaeroides, contains multiple cysteines in its C-terminal region, two of which function as a redox-active dithiol/disulfide couple with an E (m) value of -325 mV at pH 7.0 in the dark. Titrations of this dithiol/disulfide couple in illuminated samples of AppA indicate that the E (m) value of this disulfide/dithiol couple is -315 mV at pH 7.0, identical to the value obtained for AppA in the dark within the combined experimental uncertainties of the two measurements. The E (m) values of AppA and PpsR demonstrate that these proteins are thermodynamically capable of electron transfer for their activity as an anti-repressor/repressor in R. sphaeroides.

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Figures

Fig. 1
Fig. 1
Amino acid sequence alignments of PpsR/CrtJ proteins.▸ The order of organisms listed from top to bottom and their respective GenBank accession numbers are: Rhodobacter sphaeroides ZP_00913090, Rhodobacter capsulatus S17813, Jannaschia sp. CCS1 YP_508106, Loktanella vestfoldensis ZP_01002308, Roseovarius sp. 217 ZP_01034603, uncultured proteobacterium AAM48684, Erythrobacter sp. NAP1 ZP_01041674, gamma proteobacterium KT 71 EAQ96254, Bradyrhizobium sp. ORS278 AAT78846, Bradyrhizobium sp. ORS278 AAL68701, Rhodospirillum centenum ORF 933 (unpublished), Rubrivivax gelatinosus AAO93134, Rhodopseudomonas palustris YP_570870, Rhodospirillum rubrum YP_425717, Thiocapsa roseopersicina AAX53585, Thiocapsa roseopersicina AAX53580. Peptide sequences were aligned with ClustalW, using blosum matrix. All cysteines are highlighted in blue, 100% conserved residues in red, 80% conserved residues in orange, and 60% conserved residues in yellow
Fig. 2
Fig. 2
Oxidation–reduction properties of R. sphaeroides PpsR. (A) Oxidation–reduction titration of PpsR at pH 7.0. The open circles represent data obtained from mBBr titrations and the solid line represents the computer best-fit to this data based on the Nernst equation for a two-electron couple. The total DTT concentration was 1.0 mM in 100 mM HEPES buffer at pH 7.0. The redox-equilibration time was 2 h. (B) The dependence of the Em of PpsR on pH. The solid line is best-fit straight line with a slope of −55 mV/pH, which represents the theoretical slope of −59 mV/pH characteristic of a two-electron process that is accompanied by the uptake of two protons. The vertical error brackets represent the average deviation in the Em values of measurements for which replicate measurements were carried out. Reaction conditions were the same as in (A) except with different buffers used over the following pH ranges: MES, pH 5.5–6.5; HEPES, pH 7.0; TRICINE, pH 7.5–8.0; CAPSO, pH 9.0; all at 100 mM concentration
Fig. 3
Fig. 3
Oxidation–reduction properties of R. sphaeroides AppA. (A) Oxidation–reduction titration of AppA at pH 7.0. The open circles represent data obtained from mBBr titrations under dark condition at 20°C, and the solid line represents the computer best-fit to this data base on the Nernst equation for a two-electron couple. The closed circles represent data under light condition at 5°C and the dashed line represents the computer best-fit to the Nernst equation for a two-electron couple. The total DTT concentration was 2.0 mM in 100 mM HEPES buffer at pH 7.0. The redox-equilibration time was 2 h. (B) The dependence of the Em of AppA on pH. The solid line represents the theoretical slope of −59 mV/pH characteristic of a two-electron process that is accompanied by the uptake of two protons and the dashed line represents the theoretical slope of –29.5 mV/pH characteristic of a two-electron process that is accompanied by the uptake of one proton. The vertical error brackets represent the average deviation in the Em values of measurements for which replicate measurements were carried out. Reaction conditions were the same as in (A) except with different buffers used over the following pH ranges: MES, pH 5.5–6.5; HEPES, pH 7.0; TRICINE, pH 7.5–8.0; CAPSO, pH 9.0; all at 100 mM concentration
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References

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