The high-potential iron sulfur protein (HiPIP), an electron transfer mediator in photosynthetic bacteria, is a small soluble metalloprotein harboring a single iron-sulfur (Fe4S4) cluster in its molecular center. HiPIP has a high redox potential (E0 ≥ +350 mV) comparing to other protein-containing Fe4S4 clusters such as ferredoxin which has an E0 ≈ –400 mV [1,2]. This high redox potential is achieved by the transition between the resting (reduced) [Fe4S4]2+ and active (oxidized) [Fe4S4]3+ states which is governed by the hydrogen-bonding network within the local vicinity of the cluster [3]. However, resolving such atomic details with the correct geometry of the metal cluster are often hampered by X-ray radiation damage [4]. Here, we report the first radiation-damage free structures of the redox states of HiPIP at ~1.65 Å resolution using room-temperature serial femtosecond crystallography (SFX) with X-ray pulses shorter than 25 fs at megahertz intra-train repetition rates. The results reveal that most of the Fe–S/Fe–Cysteine(S) intra-atomic distances are shortened by 0.03–0.1 Å in comparison with the synchrotron models indicating that the SFX models are less affected by X-ray radiations. Our SFX models will provide an exemplar for metalloproteins enabling further theoretical and experimental studies on redox enzymology.