Thermotolerant photodependent hydrogen production by platinized photosystem i reaction centers and recombinant cytochrome c₅₅₃ from Thermosynechococcus elongatus

Photosystem I (PSI) from plants, algae, and cyanobacteria has been shown to mediate hydrogen evolution both in vivo and in vitro. We observed that metallic platinum could be photo-precipitated at the reducing end of PSI according to the reaction, [PtCl₆]^-2 + 4e^- + hν ⇒ Pt↓ + 6Cl^-. This platinum nanocluster can then receive the photo-generated electrons from PSI and function as a nanocatalyst for the evolution of molecular hydrogen in vitro. In this biomimetic reaction, sodium ascorbate is present as a sacrifical source of reducing equivalents with cyt c₅₅₃ acting as a relay between ascorbate and the lumenal side of PSI. To enable a more thermostable and kinetically optimized system for hydrogen evolution in this biomimetic photosynthetic reaction, we have begun characterizing the hydrogen evolution capabilities of trimeric PSI isolated from the thermophilic cyanobacterium, Thermosynechococcus elongatus (T. elongatus). This organism utilizes only c-type cytochrome as the primary electron donor to P₇₀₀. An expression system for T. elongatus cyt c₅₅₃ in E. coli that involves the concurrent over-expression of the entire E. coli Type I heme insertion pathway (ccmA-H) plus ALA supplementation of minimal, synthetic media have been developed and optimized. CD spectroscopy has shown that cyt c₅₅₃ is thermally stable up to a temperature of >750C. Also CD spectroscopy of PSI from T. elogatus suggests that most of the chlorophyll molecules maintain their structure until ∼67° and Chl a, the key pigment that serves as an electron donor called the special pair, is still intact up to 95°C. We have demonstrated that a Cyt-PSI-Pt system based on this cytochrome is capable of evolving hydrogen in a cyt c₅₅₃- and lightdependent fashion. Hydrogen evolution increases with temperature up to 55°C. In addition, stability studies have shown the hydrogen evolution to be stable for >80 days. We have also demonstrated through a sequence-of-addition reaction that although sodium ascorbate can directly reduce P₇₀₀ during photo-dependent hydrogen production, these experiments unequivocally show that cyt c₅₅₃ acts as a catalyst in this reaction.