His64(E7)–&gt;Tyr apomyoglobin as a reagent for measuring rates of hemin dissociation.

Hargrove, Mark S.; Singleton, Eileen W.; Quillin, M.L.; Ortiz, L.A.; Phillips, George N.; Olson, John S.; Mathews, Antony J.

doi:10.1016/s0021-9258(17)41764-9

Cited by 207 publications

(123 citation statements)

References 32 publications

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“…Mutants of apo-IsdH and apo-Mb(H64Y/V68F) were prepared as previously reported [44–46]. Briefly, 10 mL LB supplemented with 50 µg/mL kanamycin sulfate (LB/kan) was inoculated with E. coli BL21(DE3) cells bearing mutant pSUMO-IsdH or pSUMO-apoMb(H64Y/V68F) plasmid and grown overnight at 37°C with 200 rpm shaking.…”

Section: Methodsmentioning

confidence: 99%

Directed inter-domain motions enable the IsdH Staphylococcus aureus receptor to rapidly extract heme from human hemoglobin

Clayton

Ellis-Guardiola

Mahoney

et al. 2021

Preprint

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Pathogenic Staphylococcus aureus actively acquires iron from human hemoglobin (Hb) using the IsdH surface receptor. Heme extraction is mediated by a tridomain unit within the receptor that contains its second (N2) and third (N3) NEAT domains joined by a helical linker domain. Extraction occurs within a dynamic complex, in which receptors engage each globin chain; the N2 domain tightly binds to Hb, while substantial inter-domain motions within the receptor enable its N3 domain to transiently distort the globin's heme pocket. Using molecular simulations, Markov modeling, and quantitative measurements of heme transfer kinetics, we show that directed inter-domain motions within the receptor play a critical role in the extraction process. The directionality of N3 domain motion and the rate of heme extraction is controlled by amino acids within a short, flexible inter-domain tether that connects the N2 and linker domains. In the wild-type receptor directed motions originating from the tether enable the N3 domain to populate configurations capable of distorting Hb's pocket, whereas mutant receptors containing altered tethers are less able to adopt these conformers and capture heme slowly via indirect processes in which Hb first releases heme into the solvent. Thus, our results show inter-domain motions within the IsdH receptor play a critical role in its ability to extract heme from Hb and highlight the importance of directed motions by the short, unstructured, amino acid sequence connecting the domains in controlling the directionality and magnitude of these functionally important motions.

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Section: Methodsmentioning

confidence: 99%

Directed inter-domain motions enable the IsdH Staphylococcus aureus receptor to rapidly extract heme from human hemoglobin

Clayton

Ellis-Guardiola

Mahoney

et al. 2021

Preprint

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“…Absorbance changes were monitored after mixing the double-mutant apomyoglobin (apoMb) (heme acceptor) (H64Y/V86F) with samples of met form of each mutant. The apoMb reagent binds heme released from ferric Hb generating a unique spectrum for the 'green' holoMb end product as described previously [13]. Absorbance spectra between 350 and 700 nm were recorded every 2 min for 16 h at 37°C using 200 mM potassium phosphate buffer at pH 7.0 to which 600 mM sucrose was added to prevent denaturation of the resultant apoHb.…”

Section: Kinetics Of Heme Loss From Ferric Mutant Hemoglobinsmentioning

confidence: 99%

“…The final concentration of ferric Hb in heme equivalents was 2 μM, and the final concentration of H64Y/V86F apoMb was 20 μM in a 1-ml reaction volume. Data collection started immediately after mixing the two solutions; absorbance changes at 410 nm were used for the calculation of the extent of heme transfer [13].…”

Section: Kinetics Of Heme Loss From Ferric Mutant Hemoglobinsmentioning

confidence: 99%

“…Oxidative pathways described above ultimately lead to unfolding of the protein and loss of heme. To assess the rate of heme release from these mutants, we measured the absorbance changes when a heme acceptor, the doublemutant (H64Y/V86F) apomyoglobin (ApoMb), as it binds the heme released from the ferric forms of HbS and HbS mutants (Figure 5(A)) [13]. Time courses for heme loss measuring the decrease in λ 410 nm as heme transferred to the H64Y/V68F apoMb reagent is shown in Figure 5(B).…”

Section: Heme Loss From the Hbs Mutantsmentioning

confidence: 99%

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Effects of α subunit substitutions on the oxidation of βCys93 and the stability of sickle cell hemoglobin

Hicks

Meng²,

Kassa

et al. 2020

Redox Report

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The β subunit substitutions, F41Y and K82D, in sickle cell hemoglobin (Hb) (βE6 V) provides significant resistance to oxidative stress by shielding βCys93 from the oxidizing ferryl heme. We evaluated the oxidative resistance of βCys93 to hydrogen peroxide (H 2 O 2 ) in α subunit mutations in βE6 V (at both the putative and lateral contact regions) that included (1) αH20Q/βE6 V; (2) αH50Q/βE6 V; (3) αH20Q/H50Q/βE6 V; (4) αH20R/βE6 V; and (5) αH20R/H50Q/βE6 V. Estimation by mass spectrometry of irreversible oxidation of βCys93 to cysteic acid (CA) was unchanged or moderately increased in the single mutants harboring a H20Q or H50Q substitution when compared to control (βE6 V). The introduction of Arg (R) singularly or in combination with Q enhanced the pseudoperoxidative cycle by slightly decreasing the ferryl in favor of ferrous and ferric species after treatment with H 2 O 2 . Higher rates for heme loss from the ferric forms of the Q species to the receptor high affinity recombinant apomyglobin were observed in contrast to the R mutants and control. Because of their improved solubility, a combination of Q and R substitutions together with mutations carrying redox active variants (F41Y/K82D) may provide dual antioxidant and antisickling targets in the design of gene therapy-based candidates.

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“…It is noteworthy in the context of the characteristics of heme and hemoglobin binding by GAPDH, that S. gordonii is able to promote the conversion of oxyhemoglobin into methemoglobin via the action of hydrogen peroxide [32]. When in the methemoglobin form, the affinity for its ferric heme is much reduced [33] and which can more easily be sequestered by serum albumin and by the HmuY hemophore-like heme-binding protein of P. gingivalis [34]. Thus, it is likely that GAPDH may play an important role in binding free heme and in the extraction of this cofactor from other hemoproteins.…”

Section: Introductionmentioning

confidence: 99%

Porphyromonas gingivalis HmuY and Streptococcus gordonii GAPDH—Novel Heme Acquisition Strategy in the Oral Microbiome

Ślęzak

Śmiga

Smalley

et al. 2020

IJMS

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The oral cavity of healthy individuals is inhabited by commensals, with species of Streptococcus being the most abundant and prevalent in sites not affected by periodontal diseases. The development of chronic periodontitis is linked with the environmental shift in the oral microbiome, leading to the domination of periodontopathogens. Structure-function studies showed that Streptococcus gordonii employs a “moonlighting” protein glyceraldehyde-3-phosphate dehydrogenase (SgGAPDH) to bind heme, thus forming a heme reservoir for exchange with other proteins. Secreted or surface-associated SgGAPDH coordinates Fe(III)heme using His43. Hemophore-like heme-binding proteins of Porphyromonas gingivalis (HmuY), Prevotella intermedia (PinO) and Tannerella forsythia (Tfo) sequester heme complexed to SgGAPDH. Co-culturing of P. gingivalis with S. gordonii results in increased hmuY gene expression, indicating that HmuY might be required for efficient inter-bacterial interactions. In contrast to the ΔhmuY mutant strain, the wild type strain acquires heme and forms deeper biofilm structures on blood agar plates pre-grown with S. gordonii. Therefore, our novel paradigm of heme acquisition used by P. gingivalis appears to extend to co-infections with other oral bacteria and offers a mechanism for the ability of periodontopathogens to obtain sufficient heme in the host environment. Importantly, P. gingivalis is advantaged in terms of acquiring heme, which is vital for its growth survival and virulence.

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His64(E7)–>Tyr apomyoglobin as a reagent for measuring rates of hemin dissociation.

Cited by 207 publications

References 32 publications

Directed inter-domain motions enable the IsdH Staphylococcus aureus receptor to rapidly extract heme from human hemoglobin

Directed inter-domain motions enable the IsdH Staphylococcus aureus receptor to rapidly extract heme from human hemoglobin

Effects of α subunit substitutions on the oxidation of βCys93 and the stability of sickle cell hemoglobin

Porphyromonas gingivalis HmuY and Streptococcus gordonii GAPDH—Novel Heme Acquisition Strategy in the Oral Microbiome

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