Article pubs.acs.org/biochemistry
Redox-Dependent Dynamics in Heme-Bound Bacterial Iron Response Regulator (Irr) Protein Kazuo Kobayashi,*,† Megumi Nakagaki,‡ Haruto Ishikawa,‡,∇ Kazuhiro Iwai,§ Mark R. O’Brian,∥ and Koichiro Ishimori‡,⊥ †
The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan § Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Kyoto 606-8561, Japan ∥ Department of Microbiology and Immunology, State University of New York at Buffalo, Buffalo, New York 14214, United States ⊥ Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan ‡
ABSTRACT: The iron response regulator (Irr) protein from Bradyrhizobium japonicum mediates iron-dependent regulation of heme biosynthesis. Irr degrades in response to heme availability through a process that involves the binding of heme to Cys-29 in the heme regulatory motif (HRM) in the presence of molecular oxygen. In this work, we assessed the dynamics of one-electron reduction of heme-bound Irr by monitoring the formation of transient intermediates by pulse radiolysis. Hydrated electrons generated by pulse radiolysis reduced heme iron-bound Irr, facilitating the binding of molecular oxygen to the heme iron in Irr through an initial intermediate with an absorption maximum at 420 nm. This initial intermediate was converted to a secondary intermediate with an absorption maximum at 425 nm, with a first-order rate constant of 1.0 × 104 s−1. The Cys-29 → Ala (C29A) mutant of Irr, on the other hand, did not undergo the secondary phase, implying that ligand exchange of Cys-29 for another ligand takes place during the process. Spectral changes during the reduction of the heme-bound Irr revealed that binding of CO to ferrous heme consisted of two phases with kon values of 1.3 × 105 and 2.5 × 104 M−1 s−1, a finding consistent with the presence of two distinct hemes in Irr. In aerobic solutions, by contrast, oxidation of the ferrous heme to the ferric form was found to be a two-phase process. The C29A mutant was similarly oxidized, but this occurred as a single-phase process. We speculate that a reactive oxygen species essential for degradation of the protein is generated during the oxidation process.
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regulatory protein involved in iron metabolism in the mammalian system.8 Thus, the regulatory functions of heme and heme-dependent protein degradation are common to a wide variety of regulatory systems. Purified recombinant Irr has two sites that bind ferric heme with different affinities.9 One of these sites, Cys-29, lies within the HRM; the second site is a six-coodinated His/His located outside of the HRM region.9 Spectral analyses of truncation and substitution mutants of Irr have revealed that the ferrous and ferric states of heme bind to different residues.10 This suggests that redox-dependent ligand switching occurs, similar to that in the CO-sensing transcription factor CooA,11−13 the oxygen sensor EcDOS from Escherichia coli,14 cytochrome cd1 nitrite reductase,15,16 and cytochrome c maturation protein.17 In CooA, ligand switching between Cys and His, which is
he iron response regulator (Irr) protein of the bacterium Bradyrhizobium japonicum regulates the heme biosynthetic pathway, preventing the accumulation of toxic porphyrin precursors under conditions of iron limitation.1 Irr is structurally homologous to bacterial Fur,1−3 a transcriptional regulator that represses genes involved in iron metabolism. When iron is limiting, Irr protein accumulates in cells and negatively regulates heme biosynthesis by downregulating the expression of hemA and hemB, which encode the heme biosynthetic enzymes δ-aminolevulinic acid (ALA) synthase and ALA dehydratase, respectively. Upon addition of iron to the cell medium, Irr rapidly degrades, allowing depression of heme synthesis.4 The degradation of Irr is heme-dependent and involves the binding of heme to Irr3 near the N-terminus in a region termed the heme regulatory motif (HRM).5 Thus, heme acts as a sensor of iron availability for heme synthesis by regulating the degradation of Irr.6,7 It is now known that B. japonicum senses iron through heme status to control iron homeostasis in an Irr-dependent manner.7 Binding of heme to the HRM has also been found to mediate degradation of the © 2016 American Chemical Society
Received: May 20, 2016 Revised: July 1, 2016 Published: July 5, 2016 4047
DOI: 10.1021/acs.biochem.6b00512 Biochemistry 2016, 55, 4047−4054
Article
Biochemistry responsible for the physiological function of CooA,11−13 occurs within milliseconds.13 Degradation of Irr is drastically retarded in an HRMdefective mutant,4,10 implicating the binding of heme to the HRM in triggering Irr degradation. Recently, we proposed a novel oxidative modification mechanism at the HRM site and His-cluster region, as shown in Figure 1.9,18 According to this
terminal PreScission protease recognition site,9 was subsequently cloned into pGEX-6p-1 and cloned into pMal-c2X. The Irr C29A mutant was obtained by site-directed mutagenesis using a two-step PCR procedure. The nucleotide sequence of the MBP gene was confirmed by direct sequencing. BL21(DE3) (plysS) cells were transformed with the resulting plasmid and grown to saturation. A 60 mL aliquot of this culture was then mixed with 2 L of TY medium containing 50 mg/L ampicillin and incubated at 37 °C. After cultures had reached an optical density at 600 nm (OD600) of 0.6−0.8, protein expression was induced by incubating cells with 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG) for 3 h. The cells were then harvested and stored at −20 °C. Purification of Irr. The cell suspension was thawed and incubated in standard buffer [20 mM Tris-HCl (pH 7.4), 200 mM NaCl, and 1 mM EDTA] containing 15 mg/L egg white lysozyme, 20 mg/L DNase, 20 mg/L RNase, and 0.1% TritonX. After 60 min at 4 °C, the cell debris was removed by centrifugation at 18000g for 30 min. The supernatant was applied to an amylose resin (New England Biolabs) column previously equilibrated with standard buffer, and bound protein was eluted with standard buffer containing 10 mM maltose. The eluate was concentrated to 1.5 mL and then diluted in PreScission buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 10 mM EDTA]. PreScission protease (1.5 μL) was added to 1.5 mL of Irr-MBP containing 1.5 mM dithiothreitol and the mixture allowed to stand for 12 h at 4 °C. Cleaved MBP and PreScission protease were removed using a Ni-NTA column, which has a high affinity for Irr. The sample was then loaded onto a His Trap chelating column (GE Healthcare Life Sciences) equilibrated with 50 mM potassium phosphate buffer (pH 7.4) containing 500 mM NaCl and 40 mM imidazole. Irr was eluted with the same buffer containing 500 mM imidazole. The Irr thus obtained was more than 80% pure as judged by sodium dodecyl sulfate−polyacrylamide gel electrophoresis staining with Coomassie Blue R-250. The eluted fractions were dialyzed against 50 mM potassium phosphate buffer (pH 7.4). Heme-bound Irr was prepared by adding an equivalent amount of hemin dissolved in dimethylformamide to the Irr solution.9 Residual free hemin was removed from the solution, passing the heme-bound Irr through a PD-10 column (GE Healthcare). The absorption spectra of heme-bound wild-type and C29A Irr proteins revealed absorption maxima at 372 and 414 nm in the wild type and 413 nm in the C29A mutant. All hemes added to the sample were bound to Irr. Pulse Radiolysis. Pulse radiolysis experiments were performed with a pulse width of 8 ns and an energy of 27 MeV using a linear accelerator at the Institute of Scientific and Industrial Research at Osaka University.13,16,19−24 The sample was placed in a quartz cell with an optical path length of 1 cm, and the temperature of the sample was maintained at 25 °C. The light source for the spectrophotometer was a 200 W Xe lamp. After passing through an optical path, the transmitted light was analyzed and monitored using a fast spectrophotometric system composed of a Nikon monochromator, an R-928 photomultiplier, and a Unisoku data analysis system. All data points at different wavelengths were obtained in separate measurements. The concentration of hydrated electrons formed in the sample solution varied slightly between pulses (
