Cryogenic electron microscopy studies on hydrogenases

Hydrogenases are enzymes that produce or consume hydrogen (H2). They have central roles in microbial metabolism and have been of biotechnological interest. In this thesis two are studied: the putatively bifurcating hydrogenase from Thermotoga maritima (TmHydABC) and the oxygen-tolerant hydrogenase from E. coli (Hyd-1). Structural studies of the apo-TmHydABC enzyme with cryogenic electron microscopy (cryo-EM) reveal its multimetric structure and how the active sites are connected. A deeper analysis of the single-particle data with symmetry expansion shows a bridge domain that changes conformation, which is most likely mechanistically relevant. Further studies to find the ferredoxin-bound and the holo-enzyme structure were attempted but were of limited success. These structural studies in combination with theoretical thermodynamic considerations are used to propose that TmHydABC only couples reactions kinetically and not thermodynamically. A new purification strategy of Hyd-1 was developed. A his-tag is swapped for a strep-tag and placed in a less destabilizing position, using recently developed CRISPR-cas9 genetic methods for homologous recombination. The resulting preparation is homogenous and contains all three subunits: HyaA, B, and C. This preparation was then subjected to cryo-EM analysis but a severe preferred orientation problem precluded a high-resolution 3D structure. Attempts to get around this problem are presented. Finally, the hydrogenase field as a whole is critically evaluated in terms of real-world relevance and found to be of limited commercial potential, at least in terms of the hydrogen economy.