The Iron Chaperone Protein CyaY from <i>Vibrio cholerae</i> Is a Heme-Binding Protein

CyaY is an iron transport protein for iron–sulfur (Fe–S) cluster biosynthetic systems. It also transports iron to ferrochelatase that catalyzes insertion of Fe²⁺ into protoporphyrin IX. Here, we find 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 binding of heme 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

A Dye-Decolorizing Peroxidase from <i>Vibrio cholerae</i>

The dye-decolorizing peroxidase (DyP) protein from <i>Vibrio cholerae</i> (<i>Vc</i>DyP) was expressed in <i>Escherichia coli</i>, and its DyP activity was assayed by monitoring degradation of a typical anthraquinone dye, reactive blue 19 (RB19). Its kinetic activity was obtained by fitting the data to the Michaelis–Menten equation, giving <i>k</i>_cat and <i>K</i>_m values of 1.3 ± 0.3 s^–1 and 50 ± 20 μM, respectively, which are comparable to those of other DyP enzymes. The enzymatic activity of <i>Vc</i>DyP was highest at pH 4. A mutational study showed that two distal residues, Asp144 and Arg230, which are conserved in a DyP family, are essential for the DyP reaction. The crystal structure and resonance Raman spectra of <i>Vc</i>DyP indicate the transfer of a radical from heme to the protein surface, which was supported by the formation of the intermolecular covalent bond in the reaction with H₂O₂. To identify the radical site, each of nine tyrosine or two tryptophan residues was substituted. It was clarified that Tyr129 and Tyr235 are in the active site of the dye degradation reaction at lower pH, while Tyr109 and Tyr133 are the sites of an intermolecular covalent bond at higher pH. <i>Vc</i>DyP degrades RB19 at lower pH, while it loses activity under neutral or alkaline conditions because of a change in the radical transfer pathway. This finding suggests the presence of a pH-dependent switch of the radical transfer pathway, probably including His178. Although the physiological function of the DyP reaction is unclear, our findings suggest that <i>Vc</i>DyP enhances the DyP activity to survive only when it is placed under a severe condition such as being in gastric acid

Heme Proximal Hydrogen Bonding between His170 and Asp132 Plays an Essential Role in the Heme Degradation Reaction of HutZ from <i>Vibrio cholerae</i>

HutZ from <i>Vibrio cholerae</i> is an enzyme that catalyzes the oxygen-dependent degradation of heme. The crystal structure of the homologous protein from <i>Helicobacter pylori</i>, 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 H₂O₂ 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 (<i>K</i>_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 <i>K</i>_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

Cytoplasmic Heme-Binding Protein (HutX) from <i>Vibrio cholerae</i> Is an Intracellular Heme Transport Protein for the Heme-Degrading Enzyme, HutZ

HutZ is a cytoplasmic heme-binding protein from <i>Vibrio cholerae</i>. Although we have previously identified HutZ as a heme-degrading enzyme [Uchida, T., et al. (2012) <i>Chem. Commun.</i> <i>48</i>, 6741–6743], the heme transport protein for HutZ remained unknown. To identify the heme transport protein for HutZ, we focused on the heme utilization operon, <i>hutWXZ</i>. To this end, we constructed an expression system for HutX in <i>Escherichia coli</i> and purified it to homogeneity. An absorption spectral analysis demonstrated that HutX binds heme with a 1:1 stoichiometry and a dissociation constant of 7.4 nM. The crystal structure of HutX displays a fold similar to that of the homologous protein, ChuX, from <i>E. coli</i> O157:H7. A structural comparison of HutX and ChuX, and resonance Raman spectra of heme-HutX, suggest that the axial ligand of the ferric heme is Tyr90. The heme bound to HutX is transferred to HutZ with biphasic dissociation kinetics of 8.3 × 10^–2 and 1.5 × 10^–2 s^–1, values distinctly larger than those for transfer from HutX to apomyoglobin. Surface plasmon resonance experiments confirmed that HutX interacts with HutZ with a dissociation constant of ∼400 μM. These results suggest that heme is transferred from HutX to HutZ via a specific protein–protein interaction. Therefore, we can conclude that HutX is a cytoplasmic heme transport protein for HutZ

Heme Binding to Porphobilinogen Deaminase from <i>Vibrio cholerae</i> Decelerates the Formation of 1‑Hydroxymethylbilane

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 <i>Vibrio cholerae</i> was expressed in <i>Escherichia coli</i> 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 the effect of heme binding on the enzymatic activity. Therefore, coordination of His227 to heme appeared to induce reorientation of the domains containing Cys105, leading to a decrease in the enzymatic activity. This is the first report indicating that the PBGD activity is controlled by heme, the final product of heme biosynthesis. This finding improves our understanding of the mechanism by which heme biosynthesis is regulated

Heat-Induced Conformational Transition Mechanism of Heat Shock Factor 1 Investigated by Tryptophan Probe

A transcriptional regulatory system called heat shock response (HSR) has been developed in eukaryotic cells to maintain proteome homeostasis under various stresses. Heat shock factor-1 (Hsf1) plays a central role in HSR, mainly by upregulating molecular chaperones as a transcription factor. Hsf1 forms a complex with chaperones and exists as a monomer in the resting state under normal conditions. However, upon heat shock, Hsf1 is activated by oligomerization. Thus, oligomerization of Hsf1 is considered an important step in HSR. However, the lack of information about Hsf1 monomer structure in the resting state, as well as the structural change via oligomerization at heat response, impeded the understanding of the thermosensing mechanism through oligomerization. In this study, we applied solution biophysical methods, including fluorescence spectroscopy, nuclear magnetic resonance, and circular dichroism spectroscopy, to investigate the heat-induced conformational transition mechanism of Hsf1 leading to oligomerization. Our study showed that Hsf1 forms an inactive closed conformation mediated by intramolecular contact between leucine zippers (LZs), in which the intermolecular contact between the LZs for oligomerization is prevented. As the temperature increases, Hsf1 changes to an open conformation, where the intramolecular LZ interaction is dissolved so that the LZs can form intermolecular contacts to form oligomers in the active form. Furthermore, since the interaction sites with molecular chaperones and nuclear transporters are also expected to be exposed in the open conformation, the conformational change to the open state can lead to understanding the regulation of Hsf1-mediated stress response through interaction with multiple cellular components

Amorphous Aggregation of Cytochrome <i>c</i> with Inherently Low Amyloidogenicity Is Characterized by the Metastability of Supersaturation and the Phase Diagram

Despite extensive studies on the folding and function of cytochrome <i>c</i>, the mechanisms underlying its aggregation remain largely unknown. We herein examined the aggregation behavior of the physiologically relevant two types of cytochrome <i>c</i>, metal-bound cytochrome <i>c</i>, and its fragment with high amyloidogenicity as predicted in alcohol/water mixtures. Although the aggregation propensity of holo cytochrome <i>c</i> was low due to high solubility, markedly unfolded apo cytochrome <i>c</i>, lacking the heme prosthetic group, strongly promoted the propensity for amorphous aggregation with increases in hydrophobicity. Silver-bound apo cytochrome <i>c</i> increased the capacity of fibrillar aggregation (i.e., protofibrils or immature fibrils) due to subtle structural changes of apo cytochrome <i>c</i> by strong binding of silver. However, mature amyloid fibrils were not detected for any of the cytochrome <i>c</i> variants or its fragment, even with extensive ultrasonication, which is a powerful amyloid inducer. These results revealed the intrinsically low amyloidogenicity of cytochrome <i>c</i>, which is beneficial for its homeostasis and function by facilitating the folding and minimizing irreversible amyloid formation. We propose that intrinsically low amyloidogenicity of cytochrome <i>c</i> is attributed to the low metastability of supersaturation. The phase diagram constructed based on solubility and aggregate type is useful for a comprehensive understanding of protein aggregation. Furthermore, amorphous aggregation, which is also viewed as a generic property of proteins, and amyloid fibrillation can be distinguished from each other by the metastability of supersaturation