The PpaA/AerR regulators of photosynthesis gene expression from anoxygenic phototrophic proteobacteria contain heme-binding SCHIC domains

Abstract

The SCHIC domain of the B12-binding domain family present in the Rhodobacter sphaeroides AppA protein binds heme and senses oxygen. Here we show that the predicted SCHIC domain PpaA/AerR regulators also bind heme and respond to oxygen in vitro, despite their low sequence identity with AppA.

FIG. 1.

(A) Domain structure of the R. sphaeroides AppA and PpaA proteins. SCHIC, sensor containing heme instead of cobalamin (23); BLUF, sensor of blue light using FAD (17); Cys, cysteine-rich domain; Arg, arginine-rich domain. Domains of unknown function are shown on a gray background. (B) Alignment of the SCHIC domains from anaerobic (top four) and aerobic anoxygenic phototrophic proteobacteria. Black shading indicates identities within the group; gray shading indicates similarities. The consensus is derived from a larger (58 sequences) alignment of the SCHIC domains.

FIG. 2.

(A) Electronic absorption spectra of the MBP fusion to the SCHIC domain of the R. sphaeroides PpaA protein, MBP-SCHIC_Rs, as purified from E. coli. Solid trace, original spectrum; dashed trace, spectrum obtained after addition of dithionite. (B) Spectra of MBP-SCHIC_Rs purified from anaerobic E. coli cells grown in the absence (solid trace) and presence (dotted trace) of 50 μM cyanocobalamin. (C) Spectra of MBP-SCHIC_Rs after reconstitution with hemin in vitro. Solid trace, original spectrum; dashed trace, spectrum obtained after reduction with dithionite (and dithionite removal by size exclusion chromatography). (D) Spectra of MBP-SCHIC_Js after reconstitution with hemin in vitro (approximately 1:1 molar ratio). Solid trace, original spectrum; dashed trace, spectrum obtained after reduction with dithionite. Insets show magnified long-wavelength regions.

FIG. 3.

Electronic absorption spectra of MBP-SCHIC_Rs before and after exposure to 100 μM oxygen (final concentration) (A) and 200 μM oxygen (B). The medium contained 5 mM dithiothreitol and a glucose-glucose oxidase-catalase system of oxygen removal (3). Black traces, deoxy, Fe²⁺ protein; red traces, protein immediately after addition of oxygen; gold traces, protein after 3 min; cyan traces, protein after 15 min. Insets show magnified long-wavelength regions. The experimental details are given in reference .