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

Uchida, Takeshi; Kobayashi, Noriyuki; Souichiro, Muneta; Ishimori, Koichiro

doi:10.1021/acs.biochem.6b01304

Cited by 10 publications

(7 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Heme-induced oligomerization has been reported in some proteins. , The oligomerization status of PBGD was next evaluated using a gel filtration column to confirm whether quaternary structural changes occurred upon heme binding. In the absence of heme, PBGD was eluted at 24.0 mL (Figure B).…”

Section: Resultsmentioning

confidence: 99%

Heme Binding to Porphobilinogen Deaminase from Vibrio cholerae Decelerates the Formation of 1-Hydroxymethylbilane

et al. 2018

Self Cite

View full text Add to dashboard Cite

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 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.

show abstract

Section: Resultsmentioning

confidence: 99%

Heme Binding to Porphobilinogen Deaminase from Vibrio cholerae Decelerates the Formation of 1-Hydroxymethylbilane

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…Although in vitro results, specially when protein-protein interactions and protein oligomerization are concerned, are not always easy to verify directly in living cells, it still should be possible to link the findings to processes observed in vivo . As noted in the introduction, one of the primary functions of CyaY is related to its involvement in the ISC assembly machinery [ 19 , 61 ], but also in heme synthesis [ 20 ], and presumably in the regulation of ISC assembly [ 21 ]. In addition, heme biding appears to induce oligomerization of the protein, although the type of the oligomers is not known.…”

Section: Discussionmentioning

confidence: 99%

“…CyaY from Bacilus subtilis has also been shown to interact with ferrochelatase, the terminal enzyme of heme synthesis that catalyzes iron insertion into protoporphyrin IX [ 20 ]. In addition, it was shown that Vibrio cholerae CyaY binds heme with dissociation constant in the nano-molar range [ 21 ], and that heme biding induced oligomerization of the protein. The authors suggest that heme binding to CyaY serves in the regulation of ISC assembly and may facilitate iron transfer to both the ISC assembly complex and to ferrochelatase.…”

Section: Introductionmentioning

confidence: 99%

SAXS and stability studies of iron-induced oligomers of bacterial frataxin CyaY

et al. 2017

View full text Add to dashboard Cite

Frataxin is a highly conserved protein found in both prokaryotes and eukaryotes. It is involved in several central functions in cells, which include iron delivery to biochemical processes, such as heme synthesis, assembly of iron-sulfur clusters (ISC), storage of surplus iron in conditions of iron overload, and repair of ISC in aconitase. Frataxin from different organisms has been shown to undergo iron-dependent oligomerization. At least two different classes of oligomers, with different modes of oligomer packing and stabilization, have been identified. Here, we continue our efforts to explore the factors that control the oligomerization of frataxin from different organisms, and focus on E. coli frataxin CyaY. Using small-angle X-ray scattering (SAXS), we show that higher iron-to-protein ratios lead to larger oligomeric species, and that oligomerization proceeds in a linear fashion as a results of iron oxidation. Native mass spectrometry and online size-exclusion chromatography combined with SAXS show that a dimer is the most common form of CyaY in the presence of iron at atmospheric conditions. Modeling of the dimer using the SAXS data confirms the earlier proposed head-to-tail packing arrangement of monomers. This packing mode brings several conserved acidic residues into close proximity to each other, creating an environment for metal ion binding and possibly even mineralization. Together with negative-stain electron microscopy, the experiments also show that trimers, tetramers, pentamers, and presumably higher-order oligomers may exist in solution. Nano-differential scanning fluorimetry shows that the oligomers have limited stability and may easily dissociate at elevated temperatures. The factors affecting the possible oligomerization mode are discussed

show abstract

“…No pH changes in the medium were observed after the addition of hemin solution. Absorbance differences at 404-409 nm were plotted as a function of heme concentration, and apparent dissociation constants (K d,heme ) calculated using a quadratic binding equation [35].…”

Section: Measurement Of Heme Binding To Hutzmentioning

confidence: 99%

“…The inactivation of Arg92 mutants can be accounted for slow protonation of the reduced oxyferrous heme, as discussed below. The reaction mechanism of heme degradation by HutZ was previously determined [35] (Supplemental Fig. S2).…”

Section: Inactivation Mechanism Of Heme-arg92 Mutantsmentioning

confidence: 99%

Role of conserved arginine in the heme distal site of HutZ from Vibrio cholerae in the heme degradation reaction

Uchida

Dojun

Ota

et al. 2019

Archives of Biochemistry and Biophysics

Self Cite

View full text Add to dashboard Cite

HutZ from Vibrio cholerae is a dimeric enzyme that catalyzes degradation of heme. The highly conserved Arg92 residue in the HutZ family is proposed to interact with an iron-bound water molecule in the distal heme pocket. To clarify the specific role of Arg92 in the heme degradation reaction, the residue was substituted with alanine, leucine, histidine or lysine to modulate electrostatic interactions with iron-bound ligand. All four Arg92 mutants reacted with hydrogen peroxide to form verdoheme, a prominent intermediate in the heme degradation process. However, when ascorbic acid was used as an electron source, iron was not released even at pH 6.0 despite a decrease in the Soret band, indicating that non-enzymatic heme degradation occurred. Comparison of the rates of heme reduction, ligand binding and verdoheme formation suggested that proton transfer to the reduced oxyferrous heme, a potential rate-limiting step of heme degradation in HutZ, is hampered by mutation. In our previous study, we found that the increase in the distance between heme and Trp109 from 16 to 18 Å upon lowering the pH from 8.0 to 6.0 leads to activation of ascorbic acid-assisted heme degradation by HutZ. The distance in Arg92 mutants was >19 Å at pH 6.0, suggesting that subunit-subunit interactions at this pH are not suitable for heme degradation, similar to Asp132 and His63 mutants. These results suggest that interactions of Arg92 with heme-bound ligand induce alterations in the distance between subunits, which plays a key role in controlling the heme degradation activity of HutZ.

show abstract

The Iron Chaperone Protein CyaY from Vibrio cholerae Is a Heme-Binding Protein

Cited by 10 publications

References 68 publications

Heme Binding to Porphobilinogen Deaminase from Vibrio cholerae Decelerates the Formation of 1-Hydroxymethylbilane

Heme Binding to Porphobilinogen Deaminase from Vibrio cholerae Decelerates the Formation of 1-Hydroxymethylbilane

SAXS and stability studies of iron-induced oligomers of bacterial frataxin CyaY

Role of conserved arginine in the heme distal site of HutZ from Vibrio cholerae in the heme degradation reaction

Contact Info

Product

Resources

About