Inactivation of Mg chelatase during transition from anaerobic to aerobic growth in Rhodobacter capsulatus

Abstract

The facultative photosynthetic bacterium Rhodobacter capsulatus can adapt from an anaerobic photosynthetic mode of growth to aerobic heterotrophic metabolism. As this adaptation occurs, the cells must rapidly halt bacteriochlorophyll synthesis to prevent phototoxic tetrapyrroles from accumulating, while still allowing heme synthesis to continue. A likely control point is Mg chelatase, the enzyme that diverts protoporphyrin IX from heme biosynthesis toward the bacteriochlorophyll biosynthetic pathway by inserting Mg(2+) to form Mg-protoporphyrin IX. Mg chelatase is composed of three subunits that are encoded by the bchI, bchD, and bchH genes in R. capsulatus. We report that BchH is the rate-limiting component of Mg chelatase activity in cell extracts. BchH binds protoporphyrin IX, and BchH that has been expressed and purified from Escherichia coli is red in color due to the bound protoporphyrin IX. Recombinant BchH is rapidly inactivated by light in the presence of O(2), and the inactivation results in the formation of a covalent adduct between the protein and the bound protoporphyrin IX. When photosynthetically growing R. capsulatus cells are transferred to aerobic conditions, Mg chelatase is rapidly inactivated, and BchH is the component that is most rapidly inactivated in vivo when cells are exposed to aerobic conditions. The light- and O(2)-stimulated inactivation of BchH could account for the rapid inactivation of Mg chelatase in vivo and provide a mechanism for inhibiting the synthesis of bacteriochlorophyll during adaptation of photosynthetically grown cells to aerobic conditions while still allowing heme synthesis to occur for aerobic respiration.

FIG. 1.

In vitro inactivation of HisBchH by light under atmospheric (•) and lowered (○) O₂ concentration. The assays were done in duplicate and were monitored at 5-min intervals. Activities shown are the linear rates after the initial 5- to 10-min lag phase typically found with this reaction. Activities of control samples (containing unilluminated HisBchH) for atmospheric and low-O₂ treatments were 899 and 476 pmol h⁻¹ μg⁻¹ of BchD, respectively. Duplicate samples for all experimental points agreed to within 2%.

FIG. 2.

Absorbance and fluorescence emission spectra of illuminated HisBchH. Purified recombinant His-tagged R. capsulatus BchH protein was illuminated for the times indicated below with light from cool-white fluorescent tubes at an intensity of 240 μmol photons m⁻² s⁻¹. (A) Absorption spectra at 0 min (——), 5 min (- - -), 15 min (- - -), and 30 min ( ·  ·  ·  ·  · ) of illumination. (B) Fluorescence emission spectra at 0 min (——), 0.5 min (- - -), 2 min (- - -), 5 min ( ·  ·  ·  ·  · ), and 10 min (- · - · -) of illumination. The fluorescence excitation wavelength was 408 nm.

FIG. 3.

Mass spectra of tryptic digests of HisBchH before illumination (A) and after irradiation for 5 min with light from cool-white fluorescent tubes at an intensity of 250 μmol photons m⁻² s⁻¹ (B) and the primary structure of HisBchH (C). Underlined residues in panel C indicate peptides that were identified in the mass spectra, and the residues shown in bold type indicate peptide fragments that are strongly reduced or absent in the spectrum of irradiated HisBchH. These signals are marked with asterisks in panel A.

FIG. 4.

Immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins extracted from R. capsulatus during adaptation to aerobic conditions in the light or the dark. Immunoblots were probed with anti-HisBchD (A), anti-BchI (B), or anti-His-BchH (C). For all immunoblots, lane are identified as follows: lane 1, anaerobic control; lane 2, 5 min light; lane 3, 5 min dark; lane 4, 15 min light; lane 5, 15 min dark; lane 6, 30 min light; lane 7, 30 min dark. For immunoblot A, the rightmost lane contains 5 ng of purified recombinant HisBchD; for immunoblot B, the rightmost lane contains 5 ng of purified BchI; for immunoblot C, the leftmost lane contains 100 ng of purified recombinant His-BchH. The top immunoreactive band corresponds with BchH, while the lower band may be the CobN subunit of cobaltochelatase, which is similar to, but smaller than, BchH.

FIG. 5.

Structures of protoporphyrin IX and two photoproducts collectively named photoprotoporphyrin.