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

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.

A Glutathione Redox Effect on Photosynthetic Membrane Expression in Rhodospirillum rubrum

“…If we assume redox equilibrium between GSH/GSSG and the regulatory proteins included in Fig. 4, both RegB (E m ϭ Ϫ294 mV [32]) and PpsR (E m ϭ Ϫ320 mV [18]) would be Ͼ90% in the oxidized disulfide form over the 35-mV span of GSH/GSSG in Fig. 3.…”

Redox and Light Control the Heme-Sensing Activity of AppA

“…PpsR in R. sphaeroides controls heme, bacteriochlorophyll, and photosynthesis gene expression with the activity of the PpsR-AppA system highly regulated by numerous input signals such as light, redox, and heme (1, 7, 8). Several input signals directly affect the activity of PpsR, while others affect the ability of AppA to interact with PpsR.…”

The Critical Role of a Hydrogen Bond between Gln63 and Trp104 in the Blue-Light Sensing BLUF Domain That Controls AppA Activity

“…AppA is an antagonist of PpsR, a transcriptional repressor of photosynthetic genes, which regulates photosystem synthesis in response to oxygen concentration and bluelight intensity in the purple bacterium Rhodobacter sphaeroides. [4][5][6][7][8][9][10][11] AppA has at least two separate domains, an N-terminal flavin-binding BLUF domain and a C-terminal Cys-rich region where PpsR may be bound under dark conditions. It is suggested that the light state BLUF domain of AppA associates with the AppA C-terminal region to inhibit the interaction of AppA and PpsR.…”

“…AppA inhibits the DNA-binding activity of PpsR by two mechanisms: (1) through reduction of a specific intramolecular disulfide bond in oxidized PpsR and (2) through complex formation between reduced AppA and reduced PpsR. 9,11 Here, to observe the mutational effects for only blue-light control (but not redox control) of AppA activity, the gel mobility shift assays were carried out in the presence of dithiothreitol to reduce all dithiol/ disulfide pairs in AppA and PpsR. Under the reduced conditions, AppA makes a complex with PpsR in the dark to inhibit DNA binding.…”