Binding of naphthohydroxamic acid to horseradish peroxidase monitored by zinc mesoporphyrin fluorescence line narrowing

J. Fidy, G. R. Holtom, K. G. Paul, J. M. Vanderkooi

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Abstract

The effect of binding the aromatic hydrogen donor naphthohydroxamic acid (NHA) to Zn mesoporphyrin substituted horseradish peroxidase was studied by conventional and laser-excited steady-state and time-resolved luminescence spectroscopy. The free enzyme is characterized by a Soret band at 418 nm and a 0-0 bond at 587 nm, determined by fluorescence line narrowing. The fluorescence and phosphorescence maxima at 77 K are at 585 and 721 nm, respectively. At 20 K the fluorescence decay of the 0-0 band using vibronic excitation could be fit by using two components, 1.15-1.18 (A1 = 0.91-0.92) and 3.3-3.6 ns in the entire energy range of the 0-0 emissions; this leads to a mean value of 1.4 ns. The phosphorescence lifetime at 77 K was 22 ms, using a single-exponential fit, while at room temperature the lifetime decreases due to thermal enhancement of delayed fluorescence. The inhomogeneous distribution of 0-0 energies at cryogenic temperatures is narrow, with a width of 35 cm-1. Upon binding NHA, the band maxima all shift to higher energies: to 408 nm for the Soret band and to 577 and 708 nm for the fluorescence and phosphorescence maxima, respectively. Fluorescence line narrowing reveals that the 0-0 band is shifted to 579 nm and is now split into two bands separated by about 60 cm-1 in energy. In the cryogenic range the two bands are photoconvertible and can revert to the starting species in the dark in a temperature-dependent reaction. The two bands are themselves composed of multiple 0-0 lines that could not be resolved by the line narrowing technique. Both bands show fluorescence decay that could be fit by a single-exponential, 1.8- and 2.2-ns lifetime for the lower and higher energy forms, respectively. The spectral parameters are consistent with the view that, in the free enzyme, the Zn atom is out-of-plane and 5-coordinated. Upon addition of substrate, the ligation is changed in the direction of less coordination. The spectra are incompatible with the formation of a complex between NHA and the porphyrin, which means that it is unlikely that the aromatic substrate lies parallel and face to face with the heme. The two species for the protein-substrate complex were interpreted as being deformed structures that arise through vibronic interactions of the quasi-planar Zn mesoporphyrin. Following this interpretation, the loss of resolution in the line narrowed spectra upon substrate binding could be caused by either rapid interconversion between the deformed Zn mesoporphyrin structures or interaction with a close molecule above the plane. Finally, the results are contrasted with the literature data for Zn cytochrome c.

Original languageEnglish
Pages (from-to)4364-4370
Number of pages7
JournalJournal of Physical Chemistry
Volume95
Issue number11
Publication statusPublished - 1991

Fingerprint

Horseradish Peroxidase
Zinc
zinc
Fluorescence
fluorescence
acids
Acids
Phosphorescence
phosphorescence
Substrates
Cryogenics
Enzymes
life (durability)
enzymes
Proteins
energy
Porphyrins
Cytochromes c
zinc mesoporphyrin
Heme

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry

Cite this

Binding of naphthohydroxamic acid to horseradish peroxidase monitored by zinc mesoporphyrin fluorescence line narrowing. / Fidy, J.; Holtom, G. R.; Paul, K. G.; Vanderkooi, J. M.

In: Journal of Physical Chemistry, Vol. 95, No. 11, 1991, p. 4364-4370.

Research output: Contribution to journalArticle

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abstract = "The effect of binding the aromatic hydrogen donor naphthohydroxamic acid (NHA) to Zn mesoporphyrin substituted horseradish peroxidase was studied by conventional and laser-excited steady-state and time-resolved luminescence spectroscopy. The free enzyme is characterized by a Soret band at 418 nm and a 0-0 bond at 587 nm, determined by fluorescence line narrowing. The fluorescence and phosphorescence maxima at 77 K are at 585 and 721 nm, respectively. At 20 K the fluorescence decay of the 0-0 band using vibronic excitation could be fit by using two components, 1.15-1.18 (A1 = 0.91-0.92) and 3.3-3.6 ns in the entire energy range of the 0-0 emissions; this leads to a mean value of 1.4 ns. The phosphorescence lifetime at 77 K was 22 ms, using a single-exponential fit, while at room temperature the lifetime decreases due to thermal enhancement of delayed fluorescence. The inhomogeneous distribution of 0-0 energies at cryogenic temperatures is narrow, with a width of 35 cm-1. Upon binding NHA, the band maxima all shift to higher energies: to 408 nm for the Soret band and to 577 and 708 nm for the fluorescence and phosphorescence maxima, respectively. Fluorescence line narrowing reveals that the 0-0 band is shifted to 579 nm and is now split into two bands separated by about 60 cm-1 in energy. In the cryogenic range the two bands are photoconvertible and can revert to the starting species in the dark in a temperature-dependent reaction. The two bands are themselves composed of multiple 0-0 lines that could not be resolved by the line narrowing technique. Both bands show fluorescence decay that could be fit by a single-exponential, 1.8- and 2.2-ns lifetime for the lower and higher energy forms, respectively. The spectral parameters are consistent with the view that, in the free enzyme, the Zn atom is out-of-plane and 5-coordinated. Upon addition of substrate, the ligation is changed in the direction of less coordination. The spectra are incompatible with the formation of a complex between NHA and the porphyrin, which means that it is unlikely that the aromatic substrate lies parallel and face to face with the heme. The two species for the protein-substrate complex were interpreted as being deformed structures that arise through vibronic interactions of the quasi-planar Zn mesoporphyrin. Following this interpretation, the loss of resolution in the line narrowed spectra upon substrate binding could be caused by either rapid interconversion between the deformed Zn mesoporphyrin structures or interaction with a close molecule above the plane. Finally, the results are contrasted with the literature data for Zn cytochrome c.",
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N2 - The effect of binding the aromatic hydrogen donor naphthohydroxamic acid (NHA) to Zn mesoporphyrin substituted horseradish peroxidase was studied by conventional and laser-excited steady-state and time-resolved luminescence spectroscopy. The free enzyme is characterized by a Soret band at 418 nm and a 0-0 bond at 587 nm, determined by fluorescence line narrowing. The fluorescence and phosphorescence maxima at 77 K are at 585 and 721 nm, respectively. At 20 K the fluorescence decay of the 0-0 band using vibronic excitation could be fit by using two components, 1.15-1.18 (A1 = 0.91-0.92) and 3.3-3.6 ns in the entire energy range of the 0-0 emissions; this leads to a mean value of 1.4 ns. The phosphorescence lifetime at 77 K was 22 ms, using a single-exponential fit, while at room temperature the lifetime decreases due to thermal enhancement of delayed fluorescence. The inhomogeneous distribution of 0-0 energies at cryogenic temperatures is narrow, with a width of 35 cm-1. Upon binding NHA, the band maxima all shift to higher energies: to 408 nm for the Soret band and to 577 and 708 nm for the fluorescence and phosphorescence maxima, respectively. Fluorescence line narrowing reveals that the 0-0 band is shifted to 579 nm and is now split into two bands separated by about 60 cm-1 in energy. In the cryogenic range the two bands are photoconvertible and can revert to the starting species in the dark in a temperature-dependent reaction. The two bands are themselves composed of multiple 0-0 lines that could not be resolved by the line narrowing technique. Both bands show fluorescence decay that could be fit by a single-exponential, 1.8- and 2.2-ns lifetime for the lower and higher energy forms, respectively. The spectral parameters are consistent with the view that, in the free enzyme, the Zn atom is out-of-plane and 5-coordinated. Upon addition of substrate, the ligation is changed in the direction of less coordination. The spectra are incompatible with the formation of a complex between NHA and the porphyrin, which means that it is unlikely that the aromatic substrate lies parallel and face to face with the heme. The two species for the protein-substrate complex were interpreted as being deformed structures that arise through vibronic interactions of the quasi-planar Zn mesoporphyrin. Following this interpretation, the loss of resolution in the line narrowed spectra upon substrate binding could be caused by either rapid interconversion between the deformed Zn mesoporphyrin structures or interaction with a close molecule above the plane. Finally, the results are contrasted with the literature data for Zn cytochrome c.

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