Search

Neutron crystallography of proteins requires much larger crystals than those for X-ray crystallography. Obtaining large crystals of an enzyme–substrate complex is especially difficult because strict control of the stoichiometry is needed for crystal growth. Herein is reported a procedure for obtaining crystals large enough for neutron structural analysis of a bilin reductase PcyA I86D mutant complexed with its substrate, biliverdin, an open tetrapyrrol pigment. The enzyme/substrate ratio was estimated by observing changes in absorption spectra and compared to the theoretical value; 1.2 times the amount of biliverdin was required in the experiment. This proper stoichiometric ratio of PcyA I86D mutant/biliverdin reproducibly gave high-quality crystals. The buffer for the reservoir solution in the sitting drop vapor diffusion method was chosen by analysis of Wilson plots of room temperature X-ray data of the crystals obtained using three different buffers. The mixing ratio of the PcyA I86D mutant/biliverdin ...

The least understood mechanism during heme degradation by the enzyme heme oxygenase (HO) is the third step of ring opening of verdoheme to biliverdin, a process which maintains iron homeostasis. In response to this mechanistic uncertainty, we launched a combined study of X-ray crystallography and theoretical QM/MM calculations, designed to elucidate the mechanism. The air-sensitive ferrous verdoheme complex of HmuO, a heme oxygenase from Corynebacterium diphtheriae, was crystallized under anaerobic conditions. Spectral analysis of the azide-bound verdoheme-HmuO complex crystals assures that the verdoheme group remains intact during the crystallization and X-ray diffraction measurement. The structure offers the first solid evidence for the presence of a water cluster in the distal pocket of this catalytically critical intermediate. The subsequent QM/MM calculations based on this crystal structure explore the reaction mechanisms starting from the FeOOH-verdoheme and FeHOOH-verdoheme complexes, which mimic, respectively, the O(2)- and H(2)O(2)-supported degradations. In both mechanisms, the rate-determining step is the initial O-O bond breaking step, which is either homolytic (for FeHOOH-verdoheme) or coupled to electron and proton transfers (in FeOOH-verdoheme). Additionally, the calculations indicate that the FeHOOH-verdoheme complex is more reactive than the FeOOH-verdoheme complex in accord with experimental findings. QM energies with embedded MM charges are close to and yield the same conclusions as full QM/MM energies. Finally, the calculations highlight the dominant influence of the distal water cluster which acts as a biocatalyst for the conversion of verdoheme to biliverdin in the two processes, by fixing the departing OH and directing it to the requisite site of attack, and by acting as a proton shuttle and a haven for the highly reactive OH(-) nucleophile.