RESUMEN
Multiplexing of the Linac Coherent Light Source beam was demonstrated for hard X-rays by spectral division using a near-perfect diamond thin-crystal monochromator operating in the Bragg geometry. The wavefront and coherence properties of both the reflected and transmitted beams were well preserved, thus allowing simultaneous measurements at two separate instruments. In this report, the structure determination of a prototypical protein was performed using serial femtosecond crystallography simultaneously with a femtosecond time-resolved XANES studies of photoexcited spin transition dynamics in an iron spin-crossover system. The results of both experiments using the multiplexed beams are similar to those obtained separately, using a dedicated beam, with no significant differences in quality.
RESUMEN
Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized Chlorella variabilis FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer.
Asunto(s)
Carboxiliasas/química , Carboxiliasas/metabolismo , Chlorella/enzimología , Ácidos Grasos/metabolismo , Proteínas Algáceas/química , Proteínas Algáceas/metabolismo , Alcanos/metabolismo , Sustitución de Aminoácidos , Aminoácidos/metabolismo , Bicarbonatos/metabolismo , Biocatálisis , Dióxido de Carbono/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Descarboxilación , Transporte de Electrón , Flavina-Adenina Dinucleótido/química , Enlace de Hidrógeno , Luz , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxidación-Reducción , Fotones , Conformación Proteica , TemperaturaRESUMEN
Anaerobic ammonium oxidation (anammox) is a major process in the biogeochemical nitrogen cycle in which nitrite and ammonium are converted to dinitrogen gas and water through the highly reactive intermediate hydrazine. So far, it is unknown how anammox organisms convert the toxic hydrazine into nitrogen and harvest the extremely low potential electrons (-750 mV) released in this process. We report the crystal structure and cryo electron microscopy structures of the responsible enzyme, hydrazine dehydrogenase, which is a 1.7 MDa multiprotein complex containing an extended electron transfer network of 192 heme groups spanning the entire complex. This unique molecular arrangement suggests a way in which the protein stores and releases the electrons obtained from hydrazine conversion, the final step in the globally important anammox process.