RESUMEN
Heme oxygenase (HO) converts heme to carbon monoxide, biliverdin, and free iron, products that are essential in cellular redox signaling and iron recycling. In higher plants, HO is also involved in the biosynthesis of photoreceptor pigment precursors. Despite many common enzymatic reactions, the amino acid sequence identity between plant-type and other HOs is exceptionally low (â¼19.5%), and amino acids that are catalytically important in mammalian HO are not conserved in plant-type HOs. Structural characterization of plant-type HO is limited to spectroscopic characterization by electron spin resonance, and it remains unclear how the structure of plant-type HO differs from that of other HOs. Here, we have solved the crystal structure of Glycine max (soybean) HO-1 (GmHO-1) at a resolution of 1.06 Å and carried out the isothermal titration calorimetry measurements and NMR spectroscopic studies of its interaction with ferredoxin, the plant-specific electron donor. The high-resolution X-ray structure of GmHO-1 reveals several novel structural components: an additional irregularly structured region, a new water tunnel from the active site to the surface, and a hydrogen-bonding network unique to plant-type HOs. Structurally important features in other HOs, such as His ligation to the bound heme, are conserved in GmHO-1. Based on combined data from X-ray crystallography, isothermal titration calorimetry, and NMR measurements, we propose the evolutionary fine-tuning of plant-type HOs for ferredoxin dependency in order to allow adaptation to dynamic pH changes on the stroma side of the thylakoid membrane in chloroplast without losing enzymatic activity under conditions of fluctuating light.
Asunto(s)
Ferredoxinas/química , Glycine max/química , Hemo-Oxigenasa 1/química , Hemo/química , Hierro/química , Proteínas de Plantas/química , Secuencia de Aminoácidos , Biliverdina/química , Biliverdina/metabolismo , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Dominio Catalítico , Cloroplastos/química , Cloroplastos/enzimología , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Hemo/metabolismo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Enlace de Hidrógeno , Hierro/metabolismo , Simulación del Acoplamiento Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Glycine max/enzimología , Glycine max/genética , Tilacoides/química , Tilacoides/enzimologíaRESUMEN
Nitric oxide (NO) is an endogenous signaling molecule used in multiple biochemical processes. The development of switchable NO donors that deliver an NO payload under spatiotemporal control harbors many medicinal benefits. Previously, 4-fluorofuroxans were found to function as a UV light-induced NO donor under physiological conditions based on the photoinduced isomerization mechanism; however, the isomerization of fluorofuroxans with longer wavelength light is desired for further application into living systems. Herein, we report the use of photosensitizers in the photochemical isomerization of fluorofuroxan, enabling the use of visible light to induce isomerization. Among the tried photosensitizers, anthraquinone derivatives showed a good sensitizing ability to isomerize 4-fluorofuroxan to 3-fluorofuroxan using visible light. This new phenomenon was applied to the synthesis of a water-soluble anthraquinone-fluorofuroxan all-in-one molecule, which demonstrated promising NO-releasing ability using 400-500 nm irradiation. A high level of control is displayed with "on" and "off" NO-release functionality suggesting that photosensitizer-furoxan hybrids would make valuable donors. Furthermore, unlike most furoxans, NO is released in the absence of thiol cofactor.
