Examining photoinduced energy transfer in Pseudomonas aeruginosa azurin

Pseudomonas aeruginosa azurin has been an important model system for investigating fundamental electron transfer (EleT) in proteins. Early pioneering studies used ruthenium photosensitizers to induce EleT in azurin and this experimental data continues to be used to develop theories for EleT mediated through a protein matrix. In this study we show that putative EleT rates in the P. aeruginosa azurin model system, measured via photoinduced methods, can also be explained by an alternate energy transfer (EngT) mechanism. Investigation of EngT in azurin, conducted in this study, isolates and resolves confounding phenomena--i.e., zinc contamination and excited state emission--that can lead to erroneous kinetic assignments. Here we employ two azurin photosensitizer systems, the previously reported Ru(2,2'-bipyridine)2(imidazole) and an unreported phototrigger, Ru(bpy)2(phen-IA), Ru(2,2'-bipyridine)2(5-iodoacetamido-1,10-phenanthroline), that has a longer lifetime, to better resolve convoluted kinetic observations and allow us to draw clear distinctions between photoinduced EngT and EleT. Extensive metal analysis, in addition to electrochemical and photochemical (photoinduced transfer) measurements, suggests Zn-metalated azurin contamination can result in a biexponential reaction, which can be mistaken for EleT. Namely, upon photoinduction, the observed slow phase is exclusively the contribution from Zn-metalated azurin, not EleT, whereas the fast phase is the result of EngT between the photosensitizer and the Cu-site, rather than simple excited-state decay of the phototrigger.