Highly oxidized iron species play key role in the catalysis of many heme and non-heme enzymes due to their ability to activate substrates via electron or hydrogen atom transfer. Their high reactivity also makes them unstable and typically transient in nature, which hinders their structural and mechanistic investigation. Furthermore, generation of ferryloxo states typically requires addition of either oxygen and reductants or partially reduced oxygen equivalents, followed by irreversible oxygen activation. This unidirectional reaction effectively excludes investigation of associated structural changes in the protein moiety by such sensitive techniques as infrared spectroelectrochemistry, for example. Here we report on our effort to develop novel approaches toward reversible generation of ferryloxo species in horseradish peroxidase and other heme and non-heme iron proteins. We demonstrate reversible, sequential formation and quenching of Compounds I and II of HRP using several mediators in optically transparent thin layer electrochemical cell and a novel bulk optical transmission electrochemical cell. We also show that pre-conditioning of carbon-based electrodes enables generation of highly oxidized species without the use of mediators or oxygen. Following predictions of kinetically limited pre-equilibrium computational modelling and taking advantage of our chemical-free approach, we investigated the mechanisms of spontaneous quenching of highly oxidized species in HRP, the role of solvent, and the effect of pH, which yielded an unexpected result. General rules and potential pitfalls in electrochemistry of highly oxidized species in metalloenzymes will be outlined.