Regulation and function of cytochrome c' in Rhodobacter sphaeroides 2.4.3

Abstract

Cytochrome c' (Cyt c') is a c-type cytochrome with a pentacoordinate heme iron. The gene encoding this protein in Rhodobacter sphaeroides 2.4.3, designated cycP, was isolated and sequenced. Northern blot analysis and beta-galactosidase assays demonstrated that cycP transcription increased as oxygen levels decreased and was not repressed under denitrifying conditions as observed in another Rhodobacter species. CO difference spectra performed with extracts of cells grown under different conditions revealed that Cyt c' levels were highest during photosynthetic denitrifying growth conditions. The increase in Cyt c' under this condition was higher than would be predicted from transcriptional studies. Electron paramagnetic resonance analysis of whole cells demonstrated that Cyt c' binds NO during denitrification. Mass spectrometric analysis of nitrogen oxides produced by cells and purified protein did not indicate that Cyt c' has NO reductase activity. Taken together, these results suggest a model where Cyt c' in R. sphaeroides 2.4.3 may shuttle NO to the membrane, where it can be reduced.

FIG. 1.

(A) cycP region of the 2.4.3 genome. (B) lacZ fusion constructs cloned into pRK415. Color scheme: light grey, sequenced DNA; white, unsequenced DNA; dark grey, lacZ. ORF descriptions: RSP0470*, encodes a conserved hypothetical protein of unknown function; (RSP6116*, encodes a hypothetical protein of unknown function; RSP0472*, encodes a conserved hypothetical protein of unknown function; RSP0473*, encodes a hypothetical transmembrane protein similar to cardiolipin synthase/phospholipase D; cycP, encodes cytochrome c′; cybP, encodes a putative membrane-bound b-type cytochrome of unknown function. Dark box indicates location of putative FNR binding site. Restriction sites: E, EcoRI; B, BamHI.

FIG. 2.

lacZ fusion analysis of cycP transcription. (A) β-Galactosidase activity of pC′LACZ1 in 2.4.3 and R213 (FnrL⁻) backgrounds grown under various conditions. (B) β-Galactosidase activity of pC′LACZ4 in 2.4.3 and R213 (FnrL⁻) backgrounds grown under various conditions. Microaerobic/N and photosynthetic/N indicate cultures grown in media supplemented with nitrate.

FIG. 3.

Northern blot analysis using a ³²P-labeled internal fragment of cycP as a probe. Microaerobic/N and photosynthetic/N indicate cultures grown in media supplemented with nitrate. Twenty micrograms of total RNA was loaded in each lane. Unless noted, all RNA was isolated from wild-type cells. (A) RNA from cells grown under various conditions and the corresponding EtBr-stained 23S rRNA as a loading control. Numbers under each lane represent the fold change in cycP transcription relative to that in aerobic cultures. (B) Comparison of cycP transcript levels between the FnrL-deficient mutant R213, wild type, and PC12 and the corresponding EtBr-stained 23S rRNA as a loading control.

FIG. 4.

CO or NO difference spectra of purified protein and cell extracts. Spectra were obtained by scanning samples reduced with dithionite from 400 to 700 nm. Samples were then bubbled with CO or NO and scanned again. The difference spectrum for each sample was calculated by subtracting the absorbance of dithionite-reduced samples from the absorbance of CO/NO-reduced samples. (A) CO difference spectra of purified c′His; (B) CO difference spectra of concentrated protein extracts of 2.4.3 cultures grown under aerobic conditions (gray scan), PC12 grown under microaerobic conditions (solid scan), 2.4.3 grown under microaerobic conditions (dashed scan), and 2.4.3 grown under microaerobic conditions in nitrate-supplemented medium (dotted scan); (C) CO difference spectra of concentrated protein extracts of 2.4.3 grown under photosynthetic conditions in unmodified medium (dotted scan) or nitrate-supplemented medium (solid scan); (D) NO difference spectra of purified c′His.

FIG. 5.

Cyt c′ in denitrifying whole cells binds NO. (A) X-band EPR spectra of isolated whole cells of (1) Cyt c′-deficient strain grown photosynthetically with nitrate; (2) C′89 grown aerobically; (3) 2.4.3 grown photosynthetically with nitrate; (4) C′89 grown photosynthetically with nitrate. (B) Spectra of isolated whole cells of (1) C′89 grown photosynthetically with nitrate treated with 2.5 mM ferricyanide; (2) same as (1), no ferricyanide treatment; (3) Cyt c′-deficient strain, grown photosynthetically with nitrate, ferricyanide treatment; (4) C′89 grown aerobically, ferricyanide treatment; (5) 2.4.3 grown photosynthetically with nitrate, ferricyanide treatment.

FIG. 6.

Differential mass spectrometry detection of NO (m/z 30) signal after addition of cell extracts and Cyt c′. At scan 0, 66 μg of high-speed supernatant of 2.4.3 and 0.5 mM sodium nitrite was added to the DMS cell and followed until scan 100, when the electron donors ascorbate/PMS were adding, resulting in an increase in the m/z 30 signal. At scan 250, 31 μg of c′His was added, and production of the m/z 30 signal followed for another 250 scans.

FIG. 7.

Comparative effect of GSNO on growth of the Cyt c′-deficient strain PC12 and wild-type strain 2.4.3. ⧫, 2.4.3 microaerobic; ▪, 2.4.3 microaerobic plus 2 mM GSNO; ▴, cycP mutant microaerobic; x, cycP mutant microaerobic plus 2 mM GSNO. Reported values are the average for three independent experiments. Error bars represent one standard deviation.