Heme Protein Dynamics Studied by Phosphorescence of an External Phosphorescent Probe Molecule

Beckham, S.; Cook, Marcus P.; Karki, Laba; Luchsinger, M. M.; Whitlock, V. R.; Wu, Ying; Zhang, Qingfei; Schuh, Merlyn D.

doi:10.1006/abbi.1994.1190

Cited by 5 publications

(4 citation statements)

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“…As we pointed out in the introduction, understanding the mechanism by which small molecules penetrate proteins and access reactive centers is of importance. Our quenching studies coupled with the results of Schuh and associates,48–50 Barboy and Feitelson,30, 31 Papp et al,45 and Brunet et al,63 indicate that the heme group in HRP is much more protected than the heme in myoglobin and metal‐substituted hemoglobin. The remarkable finding is that the addition of the substrate analog drastically affects the penetration of oxygen, as indicated by the magnitude of the oxygen quenching rate constant.…”

Section: Discussionsupporting

confidence: 65%

“…Phosphorescence quenching profiles of BNS as a function of porphyrin‐containing substances are given in Figure 6. The quenching constant was 9.2e9 M −1 s −1 , much higher than that for native HRP at 4.6e8 M −1 s −1 49. The values for metal‐substituted proteins were also measured.…”

Section: Resultsmentioning

confidence: 93%

“…We have also employed 6‐bromo‐2‐naphthyl sulfate (BNS) as an external phosphorescent probe of heme accessibility. Schuh and collaborators introduced the use of this molecule 48–50. BNS is about the same size as the aromatic substrates of HRP and can be used to determine whether larger molecules can access the heme pocket.…”

Section: Introductionmentioning

confidence: 99%

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Accessibility of oxygen with respect to the heme pocket in horseradish peroxidase

et al. 2003

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Oxygen and other molecules of similar size take part in a variety of protein reactions. Therefore, it is critical to understand how these small molecules penetrate the protein matrix. The protein system studied in this case is horseradish peroxidase (HRP). We have converted the native HRP into a phosphorescent analog by replacing the heme prosthetic group by Pd-mesoporphyrin. Oxygen readily quenches the phosphorescence of Pd porphyrins, and this can be used to characterize oxygen diffusion through the protein matrix. Our measurements indicate that solvent viscosity and pH modulate the accessibility of the heme pocket relative to small molecules. The binding of the substrate benzohydroxamic acid (BHA) to the protein drastically impedes oxygen access to the heme pocket. These results indicate that, first, the penetration of small molecules through the protein matrix is a function of protein dynamics, and second, there are specific pathways for the diffusion of these molecules. The effect of substrate and pH on protein dynamics has been investigated with the use of molecular dynamics calculations. We demonstrate that the model of a "fluctuating entry point," as suggested by Zwanzig (J Chem Phys 1992;97:3587-3589), properly describes the diffusion of oxygen through the protein matrix.

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Section: Discussionsupporting

confidence: 65%

Section: Resultsmentioning

confidence: 93%

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Accessibility of oxygen with respect to the heme pocket in horseradish peroxidase

et al. 2003

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“…The reported energy transfer quenching rates for heme proteins are in the vicinity of 2 × 10 9 M -1 s -1 . 15 By using equation 4, the diffusion-limited bimolecular collision rate constant can be calculated. 16 (4) where N A is the Avogadro number, and R and D are the distance and diffusion constant, respectively.…”

Section: Energy Transfer To Fe(iii) Cytochrome Cmentioning

confidence: 99%

Cysteine‐Specific Blue Fluorescence Probe

Reddy

Chang

2006

J Chinese Chemical Soc

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A strong fluorescence probe has been designed to investigate cytochrome c folding kinetics. The cysteine specific modifier, 2-[(-Iodo-acetyl)-methyl-amino]-6-methylaminobenzoic acid methyl ester (1), has been prepared, and the reaction with cysteine can be performed in aqueous solution to ensure its application to protein modification. The model compound, 2-{[2-(2-Acetylamino-2-methyoxycarbonylethylsulfanyl)-acetyl]-methyl-amino}-6-methyl amino-benzoic acid methyl ester (2), resulting from the reaction of 1 with N-acetyl cysteine has emission quantum yields of 0.627 and 0.185 in nonaqueous and aqueous buffer solutions, respectively. The lifetime of 4.6 ns for compound 2 manifests a singlet excited-state. The Förster energy transfer distance of compound 2 and cytochrome c is calculated to be 45 Å.

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Dynamic quenching of porphyrin triplet states by two-photon absorbing dyes: Towards two-photon-enhanced oxygen nanosensors

Finikova

Chen

et al. 2008

Journal of Photochemistry and Photobiology A: Chemistry

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Two-photon-enhanced dendritic nanoprobes are being developed for two-photon (2P) laser scanning microscopy of oxygen [1]. In these molecular constructs, phosphorescence of metalloporphyrins is coupled to two-photon absorption (2PA) of electronically separate antenna dyes via intramolecular Förster-type resonance energy transfer (FRET). In the originally developed probes, competing electron transfer (ET) between the antennae and the long-lived triplet states of metalloporphyrins partially quenched the phosphorescence, reducing the probe's sensitivity and dynamic range. The rate of such ET can be reduced by tuning the redox potentials of the chromophores. In order to identify the optimal metalloporphyrin-2P antenna pairs, we performed screening of several phosphorescent Pt porphyrins (FRET acceptors) and 2P dyes (FRET donors) using dynamic quenching of phosphorescence. Phosphorescence lifetimes of Pt porphyrins were measured as a function of the dye concentration in organic solutions. The obtained Stern-Volmer quenching constants were correlated with the corresponding ET driving forces (ΔG ET ), calculated using the Rehm-Weller equation. FRET-pairs with minimal quenching rates were identified. The developed approach allows convenient screening of candidate-compounds for covalent assembly of 2P-enhanced triplet nanodevices. Systematic electrochemical measurements in a series of Pt porphyrins with varying peripheral substitution and conjugation pathways are presented.

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Heme Protein Dynamics Studied by Phosphorescence of an External Phosphorescent Probe Molecule

Cited by 5 publications

References 0 publications

Accessibility of oxygen with respect to the heme pocket in horseradish peroxidase

Accessibility of oxygen with respect to the heme pocket in horseradish peroxidase

Cysteine‐Specific Blue Fluorescence Probe

Dynamic quenching of porphyrin triplet states by two-photon absorbing dyes: Towards two-photon-enhanced oxygen nanosensors

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