RESUMO
We describe a simple method for the determination of heme protein reduction potentials. We use the method to determine the reduction potentials for the PAS-A domains of the regulatory heme proteins human NPAS2 (Em=-115 mV ± 2 mV, pH 7.0) and human CLOCK (Em=-111 mV ± 2 mV, pH 7.0). We suggest that the method can be easily and routinely applied to the determination of reduction potentials across the family of heme proteins.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Proteínas CLOCK/química , Heme/química , Hemeproteínas/química , Proteínas do Tecido Nervoso/química , Corantes Fluorescentes/química , Humanos , Oxazinas/química , Oxirredução , Análise EspectralRESUMO
Indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase catalyze the O(2) -dependent oxidation of l-tryptophan to N-formylkynurenine. Both are heme-containing enzymes, with a proximal histidine ligand, as found in the globins and peroxidases. From the structural information available so far, the distal heme pockets of these enzymes can contain a histidine residue (in tryptophan 2,3-dioxygenases), an arginine residue and numerous hydrophobic residues that line the pocket. We have examined the functional role of each of these residues in both human indoleamine 2,3-dioxygenase and human tryptophan 2,3-dioxygenase. We found that the distal histidine does not play an essential catalytic role, although substrate binding can be affected by removing the distal arginine and reducing the hydrophobic nature of the binding pocket. We collate the information obtained in the present study with that reported in the available literature to draw comparisons across the family and to provide a more coherent picture of how the heme pocket is optimized for tryptophan binding.
Assuntos
Hemeproteínas/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Triptofano Oxigenase/metabolismo , Triptofano/metabolismo , Domínio Catalítico , Oxirredução , Ligação Proteica , Especificidade por SubstratoRESUMO
Heme iron is often used in biology for activation of oxygen. The mechanisms of oxygen activation by heme-containing monooxygenases (the cytochrome P450s) are well known, and involve formation of a Compound I species, but information on the heme-containing dioxygenase enzymes involved in tryptophan oxidation lags far behind. In this review, we gather together information emerging recently from structural, mechanistic, spectroscopic, and computational approaches on the heme dioxygenase enzymes involved in tryptophan oxidation. We explore the subtleties that differentiate various heme enzymes from each other, and use this to piece together a developing picture for oxygen activation in this particular class of heme-containing dioxygenases.
