The cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct, two major types of DNA damage caus... more The cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct, two major types of DNA damage caused by UV light, are repaired under illumination with near UV-visible light by CPD and (6-4) photolyases, respectively. To understand the mechanism of DNA repair, we examined the resonance Raman spectra of complexes between damaged DNA and the neutral semiquinoid and oxidized forms of (6-4) and CPD photolyases. The marker band for a neutral semiquinoid flavin and band I of the oxidized flavin, which are derived from the vibrations of the benzene ring of FAD, were shifted to lower frequencies upon binding of damaged DNA by CPD photolyase but not by (6-4) photolyase, indicating that CPD interacts with the benzene ring of FAD directly but that the (6-4) photoproduct does not. Bands II and VII of the oxidized flavin and the 1398/1391 cm ؊1 bands of the neutral semiquinoid flavin, which may reflect the bending of U-shaped FAD, were altered upon substrate binding, suggesting that CPD and the (6-4) photoproduct interact with the adenine ring of FAD. When substrate was bound, there was an upshifted 1528 cm ؊1 band of the neutral semiquinoid flavin in CPD photolyase, indicating a weakened hydrogen bond at N(5)-H of FAD, and band X seemed to be downshifted in (6-4) photolyase, indicating a weakened hydrogen bond at N(3)-H of FAD. These Raman spectra led us to conclude that the two photolyases have different electron transfer mechanisms as well as different hydrogen bonding environments, which account for the higher redox potential of CPD photolyase.
Oligonucleotides used to generate mutant PAS-A-mPer2 constructs. Bases in bold indicate the mutat... more Oligonucleotides used to generate mutant PAS-A-mPer2 constructs. Bases in bold indicate the mutations introduced. Proteins Primer sequences H214A 5'-GGCCTCCATCTTTGCCTGTAAGAAGGACGCC-3' (forward) 5'-GGCGTCCTTCTTACAGGCAAAGATGGAGGCC-3' (reverse) H232A 5'-GAGTTCCTGGCTCCTGCTGACGTCAGTGTG-3' (forward) 5'-CACACTGACGTCAGCAGGAGCCAGGAACTC-3' (reverse)
HemAT from Bacillus subtilis (HemAT-Bs) is a heme-based O 2 sensor protein that acts as a signal ... more HemAT from Bacillus subtilis (HemAT-Bs) is a heme-based O 2 sensor protein that acts as a signal transducer responsible for aerotaxis. HemAT-Bs discriminates its physiological effector, O 2 , from other gas molecules to generate the aerotactic signal, but the detailed mechanism of the selective O 2 sensing is not obvious. In this study, we measured electronic absorption, electron paramagnetic resonance (EPR), and resonance Raman spectra of HemAT-Bs to elucidate the mechanism of selective O 2 sensing by HemAT-Bs. Resonance Raman spectroscopy revealed the presence of a hydrogen bond between His86 and the heme propionate only in the O 2-bound form, in addition to that between Thr95 and the hemebound O 2. The disruption of this hydrogen bond by the mutation of His86 caused the disappearance of a conformer with a direct hydrogen bond between Thr95 and the heme-bound O 2 that is present in WT HemAT-Bs. On the basis of these results, we propose a model for selective O 2 sensing by HemAT-Bs as follows. The formation of the hydrogen bond between His86 and the heme propionate induces a conformational change of the CE-loop and the E-helix by which Thr95 is located at the proper position to form the hydrogen bond with the heme-bound O 2. This stepwise conformational change would be essential to selective O 2 sensing and signal transduction by HemAT-Bs.
Synchrotron hydroxyl radical (•OH) footprinting is a technique that monitors the local changes in... more Synchrotron hydroxyl radical (•OH) footprinting is a technique that monitors the local changes in solvent accessibility of the RNA backbone on milliseconds to minutes timescales. The Mg 2þ-dependent folding of the L-21 Sca 1 Tetrahymena thermophila ribozyme has been followed using this technique at an elevated concentration of monovalent ion (200 mM NaCl) and as a function of the initial annealing conditions and substrate. Previous studies conducted at low concentrations of monovalent ion displayed sequential folding of the P4-P6 domain, the peripheral helices and the catalytic core, with each protection displaying monophasic kinetics. For ribozyme annealed in buffer containing 200 mM NaCl and folded by the addition of 10 mM MgCl 2 , multiple kinetic phases are observed for •OH protections throughout the ribozyme. The independently folding P4-P6 domain is the first to fold with its protections displaying 50-90% burst phase amplitudes. That the folding of P4-P6 within the ribozyme does not display the 100% burst phase of isolated P4-P6 at 200 mM NaCl shows that interactions with the remainder of the ribozyme impede this domain's folding. In addition, •OH protections constituting each side of a tertiary contact are not coincident in some cases, consistent with the formation of transient non-native interactions. While the peripheral contacts and triple helical scaffold exhibit substantial burst phases, the slowest protection to appear is J8/7 in the catalytic core, which displays a minimal burst amplitude and whose formation is coincident with the recovery of catalytic activity. The number of kinetic phases as well as their amplitudes and rates are different when the ribozyme is annealed in low-salt buffer and folded by the concomitant addition of monovalent and divalent cations. Annealed substrate changes the partitioning of the ribozyme among the multiple folding populations. These results provide a map of the early steps in the ribozyme's folding landscape and the degree to which the preferred pathways are dependent upon the initial reaction conditions.
We have explored the linkage of monovalent and divalent ion binding in the folding of the P4-P6 d... more We have explored the linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of Tetrahymena thermophila ribozyme by examining the Mg 2+-induced folding and the urea-induced denaturation of the folded state as a function of Na + under equilibrium folding conditions using hydroxyl radical footprinting. These studies allowed a thermodynamic examination of eight discrete protection sites within P4-P6 that are involved in several tertiary structure contacts. Monovalent ions compete with Mg 2+ ions in mediating P4-P6 folding. The urea denaturation isotherms demonstrated ∆∆G values of >2 kcal mol-1 in experiments conducted in 10 versus 200 mM NaCl at a constant 10 mM MgCl 2. However, the individual-site isotherms reported by footprinting revealed that larger than average changes in ∆G values were localized to specific sites within the Mg 2+-rich A-bulge. The competitive effects of monovalent ions were less when K + rather than Na + was the monovalent cation present. This result indicates the importance of the specific K + binding sites that are associated with AA-platform structures to P4-P6 folding and stability. These site-specific footprinting data provide quantitative and site-specific measurements of the ion-linked stability for P4-P6 that are interpreted with respect to crystallographic data.
In order to investigate the gene activation mechanism triggered by the CO binding to CooA, a heme... more In order to investigate the gene activation mechanism triggered by the CO binding to CooA, a heme-containing transcriptional activator, the heme environmental structure and the dynamics of the CO rebinding and dissociation have been examined in the absence and presence of its target DNA. In the absence of DNA, the Fe-CO and C؍O stretching Raman lines of the CO-bound CooA were observed at 487 and 1969 cm ؊1 , respectively, suggesting that a neutral histidine is an axial ligand trans to CO. The frequency of (Fe-CO) implies an open conformation of the distal heme pocket, indicating that the ligand replaced by CO is located away from the bound CO. When the target DNA was added to CO-bound CooA, an appearance of a new (Fe-CO) line at 519 cm ؊1 and narrowing of the main line at 486 cm ؊1 were observed. Although the rate of the CO dissociation was insensitive to the additions of DNA, the CO rebinding was decelerated in the presence of the target DNA, but not in the presence of nonsense DNA. These observations demonstrate the structural alterations in the heme distal site in response to binding of the target DNA and support the activation mechanism proposed for CooA, which is triggered by the movement of the heme distal ligand to modify the conformation of the DNA binding domain.
HutZ from Vibrio cholerae (VcHutZ) is a dimeric protein that catalyzes oxygen-dependent degradati... more HutZ from Vibrio cholerae (VcHutZ) is a dimeric protein that catalyzes oxygen-dependent degradation of heme. The reaction mechanism is the same as that of canonical heme oxygenase (HO), but the structure of HutZ is quite different from that of HO. Thus, we postulate that HutZ has evolved via a different pathway from that of HO. Alr5027 from cyanobacteria possessing proteins potentially related to ancestral proteins utilizing O 2 in enzymatic reactions is homologous to HutZ family proteins (67% similarity), but the heme axial ligand of HutZ is not conserved in Alr5027. To investigate whether Alr5027 can bind and degrade heme, we expressed Alr5027 in Escherichia coli and purified it. Although Alr5027 did not bind heme, replacement of Lys164, corresponding to the heme axial ligand of HutZ, with histidine conferred heme-binding capability. The K164H mutant produced verdoheme in the reaction with H 2 O 2 , indicating acquisition of heme-degradation ability. Among the mutants, the K164H mutant produced verdoheme most efficiently. Although the K164H mutant did not degrade heme through ascorbic acid, biliverdin, the final product of VcHutZ, was formed by treatment of verdoheme with ascorbic acid. An analysis of Trp103 fluorescence indicated elongation of the distance between protomers in this mutant compared with VcHutZ-the probable cause of the inefficiency of ascorbic acid-supported heme-degradation activity. Collectively, our findings indicate that a single lysine-to-histidine mutation converted Alr5027 to a heme-binding protein that can form verdoheme through H 2 O 2 , suggesting that HutZ family proteins have acquired the heme-degradation function through molecular evolution from an ancestor protein of Alr5027.
Porphobilinogen deaminase (PBGD) is an enzyme that catalyzes the formation of hydroxymethylbilane... more Porphobilinogen deaminase (PBGD) is an enzyme that catalyzes the formation of hydroxymethylbilane, a tetrapyrrole intermediate, during heme biosynthesis through the stepwise polymerization of four molecules of porphobilinogen. PBGD from Vibrio cholerae was expressed in Escherichia coli and characterized in this study. Unexpectedly, spectroscopic measurements revealed that PBGD bound one equivalent of heme with a dissociation constant of 0.33 ± 0.01 μM. The absorption and resonance Raman spectra suggested that heme is a mixture of the 5-coordinate and 6-coordinate hemes. Mutational studies indicated that the 5-coordinate heme possessed Cys105 as a heme axial ligand, and His227 was coordinated to form the 6-coordinate heme. Upon heme binding, the deamination activity decreased by approximately 15%. The crystal structure of PBGD revealed that His227 was located near Cys105, but the side chain of His227 did not point toward Cys105. The addition of the cyanide ion to heme-PBGD abolished ...
Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of... more Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of PEG. The redox potential of heme c was determined by the potentiometric titration to be +236 ± 3 mV in the absence of PEG, which was negatively shifted to +200 ± 4 mV in the presence of PEG. The underlying the rapid oxidation was explored by examining the structural changes in Cyt c in the presence of PEG using UV-visible absorption, circular dichroism, resonance Raman, and fluorescence spectroscopies. These spectroscopic analyses suggested that heme oxidation was induced by a modest tertiary structural change accompanied by a slight shift in the heme position (<1.0 Å) rather than by partial denaturation, as is observed in the presence of cardiolipin. The near-infrared spectra showed that PEG induced dehydration from Cyt c, which triggered heme displacement. The primary dehydration site was estimated to be around surface-exposed hydrophobic residues near the heme center: Ile81 and Va...
Iron regulatory proteins (IRPs), regulators of iron metabolism in mammalian cells, control the tr... more Iron regulatory proteins (IRPs), regulators of iron metabolism in mammalian cells, control the translation of proteins involved in iron uptake, storage and utilization by binding to specific iron-responsive element (IRE) sequences of mRNAs. Two homologs of IRPs (IRP1 and IRP2) have a typical heme regulatory motif (HRM), a consensus sequence found in "heme-regulated proteins". However, specific heme binding to HRM has been reported only for IRP2, which is essential for oxidative modification and loss of binding to target mRNAs. In this paper, we confirmed that IRP1 also specifically binds two molar equivalents of heme, and found that the absorption and resonance Raman spectra of heme-bound IRP1 were quite similar to those of heme-bound IRP2. This shows that the heme environmental structures in IRP1 are close to those of proteins using heme as a regulatory molecule. Pulse radiolysis experiments, however, clearly revealed an axial ligand exchange from Cys to His immediately a...
HutZ degrades heme in a manner similar to mammalian heme oxygenase, despite their low sequence an... more HutZ degrades heme in a manner similar to mammalian heme oxygenase, despite their low sequence and structural homology.
HutB is a putative heme transport protein located in the periplasmic space in Vibrio cholerae. He... more HutB is a putative heme transport protein located in the periplasmic space in Vibrio cholerae. Here, we purified HutB and characterized its heme binding properties. An analysis of the Soret band showed that there are two types of heme binding geometries depending on the heme concentration: 404-nm species are dominant at lower concentrations of heme, and 394-nm species dominate at higher concentrations. Moreover, a mutational study revealed that either Tyr65 or Tyr198 binds heme with the help of histidine, a property shared with another V. cholerae heme transport protein, HutX, despite the absence of sequence similarity, indicating that HutB acts as a heme transport protein in the periplasm.
HutZ from Vibrio cholerae is an enzyme that catalyzes the oxygen-dependent degradation of heme. T... more HutZ from Vibrio cholerae is an enzyme that catalyzes the oxygen-dependent degradation of heme. The crystal structure of the homologous protein from Helicobacter pylori, HugZ, predicts that Asp132 in HutZ is located within hydrogen-bonding distance of the heme axial ligand His170. Hydrogen bonding between His170 and Asp132 appears to be disfavored in heme-degrading enzymes, because it can contribute to the imidazolate character of the axial histidine, as observed in most heme-containing peroxidases. Thus, we investigated the role of this potential hydrogen bond in the heme degradation reaction by mutating Asp132 to Leu, Asn, or Glu and by mutating His170 to Ala. Heme degradation activity was almost completely lost in D132L and D132N mutants, whereas verdoheme formation through reaction with H 2 O 2 was comparable in the D132E mutant and wild-type enzyme. However, even at pH 6.0, when the heme is in a high-spin state, the D132E mutant was inactive toward ascorbic acid because of a significant reduction in its affinity (K d) for heme (4.1 μM) compared with that at pH 8.0 (0.027 μM). The heme degradation activity of the H170A mutant was also substantially reduced, although this mutant bound heme with a K d of 0.067 μM, despite the absence of an axial ligand. Thus, this study showed that proximal hydrogen bonding between Asp132 and His170 plays a role in retaining the heme in an appropriate position for oxygen-dependent heme degradation.
CyaY is an iron transport protein for iron-sulfur (Fe-S) cluster biosynthetic systems. It also tr... more CyaY is an iron transport protein for iron-sulfur (Fe-S) cluster biosynthetic systems. It also transports iron to ferrochelatase that catalyzes insertion of Fe 2+ into protoporphyrin IX. Here, we found that CyaY has the ability to bind heme as well as iron, exhibiting an apparent dissociation constant for heme of 21 ± 6 nM. Absorption and resonance Raman spectra revealed that both ferric and ferrous forms of heme were bound to an anionic ligand (e.g., tyrosine and/or cysteine). Consistent with this, mutagenesis studies showed that Tyr67 and Cys78 are possible heme ligands of CyaY. The binding of heme to CyaY increased the apparent dissociation constant of CyaY for iron from 65.2 to 87.9 µM. Circular dichroism spectra of CyaY suggested that heme binding to CyaY induces rearrangement of aromatic residues. Furthermore, size-exclusion column chromatography demonstrated heme-mediated oligomerization of CyaY. These results suggest that heme binding induces conformational changes, including oligomerization of CyaY, that result in a decrease in the affinity of CyaY for iron. Accordingly, the presence of excess heme in cells would lead to modulation of Fe-S cluster or heme biosynthesis. This report provides the first description of heme dependence of iron transport by CyaY.
Based on the mutational effects on the steady-state kinetics of the electron transfer reaction an... more Based on the mutational effects on the steady-state kinetics of the electron transfer reaction and our NMR analysis of the interaction site (Sakamoto, K., Kamiya, M., Imai, M., Shinzawa-Itoh, K., Uchida, T., Kawano, K., Yoshikawa, S., and Ishimori, K. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 12271-12276), we determined the structure of the electron transfer complex between cytochrome c (Cyt c) and cytochrome c oxidase (CcO) under turnover conditions and energetically characterized the interactions essential for complex formation. The complex structures predicted by the protein docking simulation were computationally selected and validated by the experimental kinetic data for mutant Cyt c in the electron transfer reaction to CcO. The interaction analysis using the selected Cyt c-CcO complex structure revealed the electrostatic and hydrophobic contributions of each amino acid residue to the free energy required for complex formation. Several charged residues showed large unfavorable ...
The cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct, two major types of DNA damage caus... more The cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct, two major types of DNA damage caused by UV light, are repaired under illumination with near UV-visible light by CPD and (6-4) photolyases, respectively. To understand the mechanism of DNA repair, we examined the resonance Raman spectra of complexes between damaged DNA and the neutral semiquinoid and oxidized forms of (6-4) and CPD photolyases. The marker band for a neutral semiquinoid flavin and band I of the oxidized flavin, which are derived from the vibrations of the benzene ring of FAD, were shifted to lower frequencies upon binding of damaged DNA by CPD photolyase but not by (6-4) photolyase, indicating that CPD interacts with the benzene ring of FAD directly but that the (6-4) photoproduct does not. Bands II and VII of the oxidized flavin and the 1398/1391 cm ؊1 bands of the neutral semiquinoid flavin, which may reflect the bending of U-shaped FAD, were altered upon substrate binding, suggesting that CPD and the (6-4) photoproduct interact with the adenine ring of FAD. When substrate was bound, there was an upshifted 1528 cm ؊1 band of the neutral semiquinoid flavin in CPD photolyase, indicating a weakened hydrogen bond at N(5)-H of FAD, and band X seemed to be downshifted in (6-4) photolyase, indicating a weakened hydrogen bond at N(3)-H of FAD. These Raman spectra led us to conclude that the two photolyases have different electron transfer mechanisms as well as different hydrogen bonding environments, which account for the higher redox potential of CPD photolyase.
Oligonucleotides used to generate mutant PAS-A-mPer2 constructs. Bases in bold indicate the mutat... more Oligonucleotides used to generate mutant PAS-A-mPer2 constructs. Bases in bold indicate the mutations introduced. Proteins Primer sequences H214A 5'-GGCCTCCATCTTTGCCTGTAAGAAGGACGCC-3' (forward) 5'-GGCGTCCTTCTTACAGGCAAAGATGGAGGCC-3' (reverse) H232A 5'-GAGTTCCTGGCTCCTGCTGACGTCAGTGTG-3' (forward) 5'-CACACTGACGTCAGCAGGAGCCAGGAACTC-3' (reverse)
HemAT from Bacillus subtilis (HemAT-Bs) is a heme-based O 2 sensor protein that acts as a signal ... more HemAT from Bacillus subtilis (HemAT-Bs) is a heme-based O 2 sensor protein that acts as a signal transducer responsible for aerotaxis. HemAT-Bs discriminates its physiological effector, O 2 , from other gas molecules to generate the aerotactic signal, but the detailed mechanism of the selective O 2 sensing is not obvious. In this study, we measured electronic absorption, electron paramagnetic resonance (EPR), and resonance Raman spectra of HemAT-Bs to elucidate the mechanism of selective O 2 sensing by HemAT-Bs. Resonance Raman spectroscopy revealed the presence of a hydrogen bond between His86 and the heme propionate only in the O 2-bound form, in addition to that between Thr95 and the hemebound O 2. The disruption of this hydrogen bond by the mutation of His86 caused the disappearance of a conformer with a direct hydrogen bond between Thr95 and the heme-bound O 2 that is present in WT HemAT-Bs. On the basis of these results, we propose a model for selective O 2 sensing by HemAT-Bs as follows. The formation of the hydrogen bond between His86 and the heme propionate induces a conformational change of the CE-loop and the E-helix by which Thr95 is located at the proper position to form the hydrogen bond with the heme-bound O 2. This stepwise conformational change would be essential to selective O 2 sensing and signal transduction by HemAT-Bs.
Synchrotron hydroxyl radical (•OH) footprinting is a technique that monitors the local changes in... more Synchrotron hydroxyl radical (•OH) footprinting is a technique that monitors the local changes in solvent accessibility of the RNA backbone on milliseconds to minutes timescales. The Mg 2þ-dependent folding of the L-21 Sca 1 Tetrahymena thermophila ribozyme has been followed using this technique at an elevated concentration of monovalent ion (200 mM NaCl) and as a function of the initial annealing conditions and substrate. Previous studies conducted at low concentrations of monovalent ion displayed sequential folding of the P4-P6 domain, the peripheral helices and the catalytic core, with each protection displaying monophasic kinetics. For ribozyme annealed in buffer containing 200 mM NaCl and folded by the addition of 10 mM MgCl 2 , multiple kinetic phases are observed for •OH protections throughout the ribozyme. The independently folding P4-P6 domain is the first to fold with its protections displaying 50-90% burst phase amplitudes. That the folding of P4-P6 within the ribozyme does not display the 100% burst phase of isolated P4-P6 at 200 mM NaCl shows that interactions with the remainder of the ribozyme impede this domain's folding. In addition, •OH protections constituting each side of a tertiary contact are not coincident in some cases, consistent with the formation of transient non-native interactions. While the peripheral contacts and triple helical scaffold exhibit substantial burst phases, the slowest protection to appear is J8/7 in the catalytic core, which displays a minimal burst amplitude and whose formation is coincident with the recovery of catalytic activity. The number of kinetic phases as well as their amplitudes and rates are different when the ribozyme is annealed in low-salt buffer and folded by the concomitant addition of monovalent and divalent cations. Annealed substrate changes the partitioning of the ribozyme among the multiple folding populations. These results provide a map of the early steps in the ribozyme's folding landscape and the degree to which the preferred pathways are dependent upon the initial reaction conditions.
We have explored the linkage of monovalent and divalent ion binding in the folding of the P4-P6 d... more We have explored the linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of Tetrahymena thermophila ribozyme by examining the Mg 2+-induced folding and the urea-induced denaturation of the folded state as a function of Na + under equilibrium folding conditions using hydroxyl radical footprinting. These studies allowed a thermodynamic examination of eight discrete protection sites within P4-P6 that are involved in several tertiary structure contacts. Monovalent ions compete with Mg 2+ ions in mediating P4-P6 folding. The urea denaturation isotherms demonstrated ∆∆G values of >2 kcal mol-1 in experiments conducted in 10 versus 200 mM NaCl at a constant 10 mM MgCl 2. However, the individual-site isotherms reported by footprinting revealed that larger than average changes in ∆G values were localized to specific sites within the Mg 2+-rich A-bulge. The competitive effects of monovalent ions were less when K + rather than Na + was the monovalent cation present. This result indicates the importance of the specific K + binding sites that are associated with AA-platform structures to P4-P6 folding and stability. These site-specific footprinting data provide quantitative and site-specific measurements of the ion-linked stability for P4-P6 that are interpreted with respect to crystallographic data.
In order to investigate the gene activation mechanism triggered by the CO binding to CooA, a heme... more In order to investigate the gene activation mechanism triggered by the CO binding to CooA, a heme-containing transcriptional activator, the heme environmental structure and the dynamics of the CO rebinding and dissociation have been examined in the absence and presence of its target DNA. In the absence of DNA, the Fe-CO and C؍O stretching Raman lines of the CO-bound CooA were observed at 487 and 1969 cm ؊1 , respectively, suggesting that a neutral histidine is an axial ligand trans to CO. The frequency of (Fe-CO) implies an open conformation of the distal heme pocket, indicating that the ligand replaced by CO is located away from the bound CO. When the target DNA was added to CO-bound CooA, an appearance of a new (Fe-CO) line at 519 cm ؊1 and narrowing of the main line at 486 cm ؊1 were observed. Although the rate of the CO dissociation was insensitive to the additions of DNA, the CO rebinding was decelerated in the presence of the target DNA, but not in the presence of nonsense DNA. These observations demonstrate the structural alterations in the heme distal site in response to binding of the target DNA and support the activation mechanism proposed for CooA, which is triggered by the movement of the heme distal ligand to modify the conformation of the DNA binding domain.
HutZ from Vibrio cholerae (VcHutZ) is a dimeric protein that catalyzes oxygen-dependent degradati... more HutZ from Vibrio cholerae (VcHutZ) is a dimeric protein that catalyzes oxygen-dependent degradation of heme. The reaction mechanism is the same as that of canonical heme oxygenase (HO), but the structure of HutZ is quite different from that of HO. Thus, we postulate that HutZ has evolved via a different pathway from that of HO. Alr5027 from cyanobacteria possessing proteins potentially related to ancestral proteins utilizing O 2 in enzymatic reactions is homologous to HutZ family proteins (67% similarity), but the heme axial ligand of HutZ is not conserved in Alr5027. To investigate whether Alr5027 can bind and degrade heme, we expressed Alr5027 in Escherichia coli and purified it. Although Alr5027 did not bind heme, replacement of Lys164, corresponding to the heme axial ligand of HutZ, with histidine conferred heme-binding capability. The K164H mutant produced verdoheme in the reaction with H 2 O 2 , indicating acquisition of heme-degradation ability. Among the mutants, the K164H mutant produced verdoheme most efficiently. Although the K164H mutant did not degrade heme through ascorbic acid, biliverdin, the final product of VcHutZ, was formed by treatment of verdoheme with ascorbic acid. An analysis of Trp103 fluorescence indicated elongation of the distance between protomers in this mutant compared with VcHutZ-the probable cause of the inefficiency of ascorbic acid-supported heme-degradation activity. Collectively, our findings indicate that a single lysine-to-histidine mutation converted Alr5027 to a heme-binding protein that can form verdoheme through H 2 O 2 , suggesting that HutZ family proteins have acquired the heme-degradation function through molecular evolution from an ancestor protein of Alr5027.
Porphobilinogen deaminase (PBGD) is an enzyme that catalyzes the formation of hydroxymethylbilane... more Porphobilinogen deaminase (PBGD) is an enzyme that catalyzes the formation of hydroxymethylbilane, a tetrapyrrole intermediate, during heme biosynthesis through the stepwise polymerization of four molecules of porphobilinogen. PBGD from Vibrio cholerae was expressed in Escherichia coli and characterized in this study. Unexpectedly, spectroscopic measurements revealed that PBGD bound one equivalent of heme with a dissociation constant of 0.33 ± 0.01 μM. The absorption and resonance Raman spectra suggested that heme is a mixture of the 5-coordinate and 6-coordinate hemes. Mutational studies indicated that the 5-coordinate heme possessed Cys105 as a heme axial ligand, and His227 was coordinated to form the 6-coordinate heme. Upon heme binding, the deamination activity decreased by approximately 15%. The crystal structure of PBGD revealed that His227 was located near Cys105, but the side chain of His227 did not point toward Cys105. The addition of the cyanide ion to heme-PBGD abolished ...
Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of... more Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of PEG. The redox potential of heme c was determined by the potentiometric titration to be +236 ± 3 mV in the absence of PEG, which was negatively shifted to +200 ± 4 mV in the presence of PEG. The underlying the rapid oxidation was explored by examining the structural changes in Cyt c in the presence of PEG using UV-visible absorption, circular dichroism, resonance Raman, and fluorescence spectroscopies. These spectroscopic analyses suggested that heme oxidation was induced by a modest tertiary structural change accompanied by a slight shift in the heme position (<1.0 Å) rather than by partial denaturation, as is observed in the presence of cardiolipin. The near-infrared spectra showed that PEG induced dehydration from Cyt c, which triggered heme displacement. The primary dehydration site was estimated to be around surface-exposed hydrophobic residues near the heme center: Ile81 and Va...
Iron regulatory proteins (IRPs), regulators of iron metabolism in mammalian cells, control the tr... more Iron regulatory proteins (IRPs), regulators of iron metabolism in mammalian cells, control the translation of proteins involved in iron uptake, storage and utilization by binding to specific iron-responsive element (IRE) sequences of mRNAs. Two homologs of IRPs (IRP1 and IRP2) have a typical heme regulatory motif (HRM), a consensus sequence found in "heme-regulated proteins". However, specific heme binding to HRM has been reported only for IRP2, which is essential for oxidative modification and loss of binding to target mRNAs. In this paper, we confirmed that IRP1 also specifically binds two molar equivalents of heme, and found that the absorption and resonance Raman spectra of heme-bound IRP1 were quite similar to those of heme-bound IRP2. This shows that the heme environmental structures in IRP1 are close to those of proteins using heme as a regulatory molecule. Pulse radiolysis experiments, however, clearly revealed an axial ligand exchange from Cys to His immediately a...
HutZ degrades heme in a manner similar to mammalian heme oxygenase, despite their low sequence an... more HutZ degrades heme in a manner similar to mammalian heme oxygenase, despite their low sequence and structural homology.
HutB is a putative heme transport protein located in the periplasmic space in Vibrio cholerae. He... more HutB is a putative heme transport protein located in the periplasmic space in Vibrio cholerae. Here, we purified HutB and characterized its heme binding properties. An analysis of the Soret band showed that there are two types of heme binding geometries depending on the heme concentration: 404-nm species are dominant at lower concentrations of heme, and 394-nm species dominate at higher concentrations. Moreover, a mutational study revealed that either Tyr65 or Tyr198 binds heme with the help of histidine, a property shared with another V. cholerae heme transport protein, HutX, despite the absence of sequence similarity, indicating that HutB acts as a heme transport protein in the periplasm.
HutZ from Vibrio cholerae is an enzyme that catalyzes the oxygen-dependent degradation of heme. T... more HutZ from Vibrio cholerae is an enzyme that catalyzes the oxygen-dependent degradation of heme. The crystal structure of the homologous protein from Helicobacter pylori, HugZ, predicts that Asp132 in HutZ is located within hydrogen-bonding distance of the heme axial ligand His170. Hydrogen bonding between His170 and Asp132 appears to be disfavored in heme-degrading enzymes, because it can contribute to the imidazolate character of the axial histidine, as observed in most heme-containing peroxidases. Thus, we investigated the role of this potential hydrogen bond in the heme degradation reaction by mutating Asp132 to Leu, Asn, or Glu and by mutating His170 to Ala. Heme degradation activity was almost completely lost in D132L and D132N mutants, whereas verdoheme formation through reaction with H 2 O 2 was comparable in the D132E mutant and wild-type enzyme. However, even at pH 6.0, when the heme is in a high-spin state, the D132E mutant was inactive toward ascorbic acid because of a significant reduction in its affinity (K d) for heme (4.1 μM) compared with that at pH 8.0 (0.027 μM). The heme degradation activity of the H170A mutant was also substantially reduced, although this mutant bound heme with a K d of 0.067 μM, despite the absence of an axial ligand. Thus, this study showed that proximal hydrogen bonding between Asp132 and His170 plays a role in retaining the heme in an appropriate position for oxygen-dependent heme degradation.
CyaY is an iron transport protein for iron-sulfur (Fe-S) cluster biosynthetic systems. It also tr... more CyaY is an iron transport protein for iron-sulfur (Fe-S) cluster biosynthetic systems. It also transports iron to ferrochelatase that catalyzes insertion of Fe 2+ into protoporphyrin IX. Here, we found that CyaY has the ability to bind heme as well as iron, exhibiting an apparent dissociation constant for heme of 21 ± 6 nM. Absorption and resonance Raman spectra revealed that both ferric and ferrous forms of heme were bound to an anionic ligand (e.g., tyrosine and/or cysteine). Consistent with this, mutagenesis studies showed that Tyr67 and Cys78 are possible heme ligands of CyaY. The binding of heme to CyaY increased the apparent dissociation constant of CyaY for iron from 65.2 to 87.9 µM. Circular dichroism spectra of CyaY suggested that heme binding to CyaY induces rearrangement of aromatic residues. Furthermore, size-exclusion column chromatography demonstrated heme-mediated oligomerization of CyaY. These results suggest that heme binding induces conformational changes, including oligomerization of CyaY, that result in a decrease in the affinity of CyaY for iron. Accordingly, the presence of excess heme in cells would lead to modulation of Fe-S cluster or heme biosynthesis. This report provides the first description of heme dependence of iron transport by CyaY.
Based on the mutational effects on the steady-state kinetics of the electron transfer reaction an... more Based on the mutational effects on the steady-state kinetics of the electron transfer reaction and our NMR analysis of the interaction site (Sakamoto, K., Kamiya, M., Imai, M., Shinzawa-Itoh, K., Uchida, T., Kawano, K., Yoshikawa, S., and Ishimori, K. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 12271-12276), we determined the structure of the electron transfer complex between cytochrome c (Cyt c) and cytochrome c oxidase (CcO) under turnover conditions and energetically characterized the interactions essential for complex formation. The complex structures predicted by the protein docking simulation were computationally selected and validated by the experimental kinetic data for mutant Cyt c in the electron transfer reaction to CcO. The interaction analysis using the selected Cyt c-CcO complex structure revealed the electrostatic and hydrophobic contributions of each amino acid residue to the free energy required for complex formation. Several charged residues showed large unfavorable ...
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Papers by Takeshi Uchida