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. 2012 Apr 20;287(17):13850-8.
doi: 10.1074/jbc.M112.346494. Epub 2012 Feb 29.

PpsR, a regulator of heme and bacteriochlorophyll biosynthesis, is a heme-sensing protein

Liang Yin  1 , Vladimira Dragnea, Carl E Bauer
Affiliations

PpsR, a regulator of heme and bacteriochlorophyll biosynthesis, is a heme-sensing protein

Liang Yin et al. J Biol Chem. .

Abstract

Heme-mediated regulation, presented in many biological processes, is achieved in part with proteins containing heme regulatory motif. In this study, we demonstrate that FLAG-tagged PpsR isolated from Rhodobacter sphaeroides cells contains bound heme. In vitro heme binding studies with tagless apo-PpsR show that PpsR binds heme at a near one-to-one ratio with a micromolar binding constant. Mutational and spectral assays suggest that both the second Per-Arnt-Sim (PAS) and DNA binding domains of PpsR are involved in the heme binding. Furthermore, we show that heme changes the DNA binding patterns of PpsR and induces different responses of photosystem genes expression. Thus, PpsR functions as both a redox and heme sensor to coordinate the amount of heme, bacteriochlorophyll, and photosystem apoprotein synthesis thereby providing fine tune control to avoid excess free tetrapyrrole accumulation.

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Figures

FIGURE 1.
FIGURE 1.
PpsR binds heme. A, UV-visible spectrum of purified PpsR-FLAG from R. sphaeroides. B, UV-visible spectrum of heme and PpsR-heme (the spectrum of PpsR is subtracted) under oxidizing conditions. 5 μm heme was incubated with 10 μm PpsR for at least 20 min before the spectrum was taken. C, UV-visible spectrum of heme and PpsR-heme under reducing conditions. D, titration of PpsR in to heme. The change of the absorbance at 280 nm indicates the change of protein concentration whereas the change of Soret peaks at 370 nm indicating the formation of PpsR-heme complex. E, binding constant of PpsR-heme interaction. Heme was titrated into 1 μm PpsR, with at least 5 min of incubation time between each step. The data were fitted with one-to-one binding model. The same settings were applied to 1 μm lysozyme.
FIGURE 2.
FIGURE 2.
Characterization of the heme-binding domain in PpsR. A, domains structure of PpsR, PpsR1–376, and PpsR1–256. B, UV-visible spectrum of heme and different PpsR constructs under oxidizing conditions. 4.6 μm heme was incubated with 9.4 μm different constructs for at least 20 min before the spectrum were taken. C, UV-visible spectrum of heme and different PpsR constructs under reducing conditions. The same samples used for oxidized spectrum were reduced with 5 mm dithionite for the reduced spectrum. D, UV-visible spectrum of PpsRH275A-heme. 4.1 μm heme was incubated with 8.2 μm PpsRH275A for at least 20 min before the spectrum were taken. E, UV-visible spectrum of PpsRC424A-heme. 4.1 μm heme was incubated with 8.2 m μm PpsRC424A for at least 20 min before the spectrum was taken.
FIGURE 3.
FIGURE 3.
Heme regulates PpsR-DNA binding. PpsR or its mutant was incubated with 2-fold of heme for 20 min before adding DNA when needed. 10 nm probe was used to bind PpsR unless noted otherwise. A, effect of heme on wild-type PpsR-puc interaction under oxidizing conditions. B, effect of heme on wild-type PpsR-puc interaction under reducing conditions. C, effect of heme on the PpsRH275A-puc interaction under oxidizing conditions. D, effect of heme on the PpsRH275A-puc interaction under reducing conditions. E, effect of heme on the PpsRC424A-puc interaction under oxidizing conditions.
FIGURE 4.
FIGURE 4.
DNase I footprinting of PpsR and puc promoter. 10 μm PpsR was incubated with puc probe before being applied to DNase I digestion. PpsR binding sites are represented with brackets. Changes in binding are indicated with shaded and dashed boxes.
FIGURE 5.
FIGURE 5.
Heme effect on the transcription levels of PpsR-regulated genes. A, PpsR-regulated photosynthesis and tetrapyrrole biosynthesis genes. The detailed diagram is described in supplemental Fig. 5. B, changes in relative transcription level of various PpsR-regulated genes in wild-type R. sphaeroides culture after addition of heme to the culture. After 20 min of starvation, 25 μm heme was added into the culture, and the samples were taken after another 20 min of incubation and normalized (supplemental Fig. 6). Data shown are mean ± S.E. (error bars). C, similar to B with the exception that mRNA levels were analyzed in the ppsR deletion strain, PPSR1.
FIGURE 6.
FIGURE 6.
Model of PpsR-heme interaction. Location of Cys424 in the HTH domain is based on structure modeled after the HTH domain of Fis. The two chains of PpsR DNA binding domain are in red and blue with Cys424 shown in green. According to a theoretical model of Fis, bent DNA would interact with PpsR at the bottom of this molecule (33).
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