Rieske dioxygenase systems (comprised of reductase, ferredoxin, and oxygenase components) are currently being used in a variety of applications ranging from drug development to cleaning environmental pollution. The diversity of applications is in part due to the multitude of reactions catalyzed by dioxygenase enzymes and the great range of reduction potentials that the Rieske [2Fe-2S] cluster can possess. Whereas some Rieske [2Fe-2S] clusters have reduction potentials as low as -150 mV, other clusters have potentials as high as +400 mV. Following the determination of the X-ray crystallographic structure of two additional Rieske dioxygenase ferredoxins, we analyzed seventeen structures to elucidate the effect of the composition and structural variation on the reduction potential of the Rieske iron-sulfur cluster. These included protein structures from bacterial Rieske dioxygenase ferredoxins, mitochondrial cytochrome bc1 complexes, and the chloroplast cytochrome b6f complex. From the structures of two dioxygenases, NDO-O9816--4 and BPDO-OB1, the binding orientations of ten aromatic substrates were calculated. From the preferred binding orientation, the regio-specificities of Rieske dioxygenase enzymes were evaluated. A novel substrate channel was identified from the superposition of sixteen distinct Rieske dioxygenase structures of NDO-O9816--4. This pathway, leading from the central, hollow core of the alpha3beta 3 enzyme into the active site, is the presumed path by which oxygen enters the active site. Computer simulations and crystallographic refinement were used to explore this possibility. Taken together, these findings strengthen our understanding of bacterial Rieske dioxygenase systems. Methods were developed to predict Rieske protein reduction potentials and Rieske dioxygenase enzymatic products. Finally, the path by which one of the substrates of dioxygenase enzymes reaches the active site was determined, which answers one of the several remaining questions in bacterial Rieske dioxygenase research.