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Use of free energy relationships to probe the individual steps of hydroxylation of p-hydroxybenzoate hydroxylase: studies with a series of 8-substituted flavins.
Biochemistry. 1999 Jun 22; 38(25):8124-37.B

Abstract

We report Hammett correlations, using 8-substituted flavins, to clarify the mechanism of hydroxylation by p-hydroxybenzoate hydroxylase (PHBH). The 8-position of the FAD isoalloxazine ring was chosen for modifications, because in PHBH it has minimal interactions with the protein, and it is accessible to solvent and away from the site of hydroxylation. Although two intermediates, a flavin-C4a-hydroperoxide and a flavin-C4a-hydroxide, are known to participate in hydroxylation, the mechanism of oxygen transfer remains controversial. Mechanisms as diverse as electrophilic aromatic substitution, diradical formation, and isoalloxazine ring opening have been proposed. In the studies reported here, it was possible to monitor spectrally each of the individual steps involved in hydroxylation, because the FAD cofactor acts as a reporter group. Thus, with PHBH, substituted separately with nine derivatives of FAD altered in the 8-position, quantitative structure-reactivity relationships (QSAR) have been applied to probe the mechanisms of formation of the flavin-C4a-hydroperoxide, the conversion to the flavin-C4a-hydroxide with concomitant oxygen transfer to the substrate, and the dehydration of the flavin-C4a-hydroxide to form oxidized FAD. The individual chemical steps in the mechanism of PHBH were not altered when using any of the modified flavins, and normal products were obtained; however, the rates of individual steps were affected, and depended on the electronic properties of the 8-substituent. Increased hydroxylation rates were observed when a more electrophilic flavin-C4a-hydroperoxide (i.e., with an electron-withdrawing substituent at the 8-position) is bound to PHBH. On the basis of QSAR analysis, we conclude that the mechanism of the hydroxylation step is best described by electrophilic aromatic substitution.

Authors+Show Affiliations

Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

10387058

Citation

Ortiz-Maldonado, M, et al. "Use of Free Energy Relationships to Probe the Individual Steps of Hydroxylation of P-hydroxybenzoate Hydroxylase: Studies With a Series of 8-substituted Flavins." Biochemistry, vol. 38, no. 25, 1999, pp. 8124-37.
Ortiz-Maldonado M, Ballou DP, Massey V. Use of free energy relationships to probe the individual steps of hydroxylation of p-hydroxybenzoate hydroxylase: studies with a series of 8-substituted flavins. Biochemistry. 1999;38(25):8124-37.
Ortiz-Maldonado, M., Ballou, D. P., & Massey, V. (1999). Use of free energy relationships to probe the individual steps of hydroxylation of p-hydroxybenzoate hydroxylase: studies with a series of 8-substituted flavins. Biochemistry, 38(25), 8124-37.
Ortiz-Maldonado M, Ballou DP, Massey V. Use of Free Energy Relationships to Probe the Individual Steps of Hydroxylation of P-hydroxybenzoate Hydroxylase: Studies With a Series of 8-substituted Flavins. Biochemistry. 1999 Jun 22;38(25):8124-37. PubMed PMID: 10387058.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Use of free energy relationships to probe the individual steps of hydroxylation of p-hydroxybenzoate hydroxylase: studies with a series of 8-substituted flavins. AU - Ortiz-Maldonado,M, AU - Ballou,D P, AU - Massey,V, PY - 1999/7/1/pubmed PY - 1999/7/1/medline PY - 1999/7/1/entrez SP - 8124 EP - 37 JF - Biochemistry JO - Biochemistry VL - 38 IS - 25 N2 - We report Hammett correlations, using 8-substituted flavins, to clarify the mechanism of hydroxylation by p-hydroxybenzoate hydroxylase (PHBH). The 8-position of the FAD isoalloxazine ring was chosen for modifications, because in PHBH it has minimal interactions with the protein, and it is accessible to solvent and away from the site of hydroxylation. Although two intermediates, a flavin-C4a-hydroperoxide and a flavin-C4a-hydroxide, are known to participate in hydroxylation, the mechanism of oxygen transfer remains controversial. Mechanisms as diverse as electrophilic aromatic substitution, diradical formation, and isoalloxazine ring opening have been proposed. In the studies reported here, it was possible to monitor spectrally each of the individual steps involved in hydroxylation, because the FAD cofactor acts as a reporter group. Thus, with PHBH, substituted separately with nine derivatives of FAD altered in the 8-position, quantitative structure-reactivity relationships (QSAR) have been applied to probe the mechanisms of formation of the flavin-C4a-hydroperoxide, the conversion to the flavin-C4a-hydroxide with concomitant oxygen transfer to the substrate, and the dehydration of the flavin-C4a-hydroxide to form oxidized FAD. The individual chemical steps in the mechanism of PHBH were not altered when using any of the modified flavins, and normal products were obtained; however, the rates of individual steps were affected, and depended on the electronic properties of the 8-substituent. Increased hydroxylation rates were observed when a more electrophilic flavin-C4a-hydroperoxide (i.e., with an electron-withdrawing substituent at the 8-position) is bound to PHBH. On the basis of QSAR analysis, we conclude that the mechanism of the hydroxylation step is best described by electrophilic aromatic substitution. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/10387058/Use_of_free_energy_relationships_to_probe_the_individual_steps_of_hydroxylation_of_p_hydroxybenzoate_hydroxylase:_studies_with_a_series_of_8_substituted_flavins_ L2 - https://doi.org/10.1021/bi990560e DB - PRIME DP - Unbound Medicine ER -