Photosystem I/dithiol molecular wire/nanoparticle bioconjugates have been shown to photocatalytically generate dihydrogen at linear rates when continuously illuminated. In an effort to maximize H2production, the pH and ionic concentration of the solution, the mobility of the electron donor, the length and degree of saturation of the molecular wire, and the intensity of the light were systematically investigated. Optimal conditions included a solution buffered at pH 6.0, cross-linked plastocyanin, rebuilt spinach PS I, and the use of 1,4-benzenedithiol to connect PS I to the Pt nanoparticle. Illumination of this optimized Photosystem I/dithiol molecular wire/nanoparticle bioconjugate at a light intensity of 70 μE m−2s−1generated a measured one time H2production rate of 312 μmol H2mg Chl−1h−1. [ABSTRACT FROM AUTHOR]