PURIFICATION AND CHARACTERIZATION OF A RECOMBINANT SMALL SUBUNIT OF THE MEMBRANE‐BOUND HYDROGENASE OF RALSTONIA EUTROPHA: TOWARD CONSTRUCTION OF A PHOTOCHEMICAL H2‐PRODUCING MODULE

Open Access
Author:
Price, Ian Robert
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • John H Golbeck, Thesis Supervisor
  • Joseph C. Reese, Honors Advisor
  • Scott Brian Selleck, Faculty Reader
Keywords:
  • photosystem I
  • molecular wire
  • hydrogenase
  • hydrogen
  • Ralstonia eutropha
  • photosynthesis
  • bioconjugate
Abstract:
In recent decades, there has been an increased interest in utilizing the process of photosynthesis to help solve humans’ energy needs, both in and ex vivo. Concurrently, interest has grown in hydrogen as a major component of the energy economy. Many methods of utilizing the high quantum yield and energy efficiency evolved in the photosynthetic complexes of cyanobacteria or plants have been proposed. One method developed in this laboratory involves the direct coupling of a photosystem I complex to an exogenous catalyst (synthetic or proteinaceous) via a so‐called “molecular wire”. This approach generally involves a modified hydrocarbon artificially inserted into the electron transport chain of photosystem on one end and attached to the catalyst on the other end. Hydrogen production via such substitution has been demonstrated in earlier modules in the Golbeck lab. Hydrogenase enzymes are clear choices for biological catalysts of the (2H+ + 2e⁻ -> H2) half reaction in the production of hydrogen. The [NiFe] hydrogenases have garnered particular interest for such use in hydrogen production due to their resistance to irreversible inactivation by oxygen (O2). [NiFe] hydrogenases are heterodimeric, with a large subunit that contains the [NiFe] active site and a small subunit that contains an electron transport chain with two [4Fe‐4S] clusters and a [3Fe‐4S] cluster. In particular, the [NiFe] hydrogenases of the Knallgas bacteria, including the genus Ralstonia, are of interest because of their ability not just to resist permanent O2 damage, but to retain their reversible H2‐oxidizing activity at atmospheric O2 levels. Here, a recombinant form of the small subunit of the membrane‐bound hydrogenase of Ralstonia eutropha has been engineered to contain an open coordination site in the distal [4Fe‐4S] cluster in the electron transport chain (His 187 converted to a Gly). Said protein has been cloned into and overexpressed in Escherichia coli, without the large subunit. Several purification schemes were evaluated to overcome the persistent insolubility of HoxK (small subunit). Electron transport‐associated iron‐sulfur clusters were reconstituted by an accepted protocol. Electron paramagnetic resonance spectroscopic analysis confirmed the formation of iron‐sulfur clusters and may suggest the recruitment of an exogenous ligand (2‐mercaptoethanol) into the missing coordination site of the distal iron‐sulfur cluster. This construction, along with reconstituted active large subunit, could constitute part of an effective photochemical module for the ex vivo synthesis of H2 via coupling to a photosystem I by means of the “molecular wire” method described. Purification methods used for the other proteins necessary for the photochemical module suggested are also provided. Synechococcus sp. PCC 7002 PsaC molecular‐wire mutant has been constructed from E. coli‐expressed protein and the Synechococcus PCC 7002 photosystem I from the native cyanobacteria.