A strenuous experimental journey searching for spectroscopic evidence of a bridging nickel–iron–hydride in [NiFe] hydrogenase

Direct spectroscopic evidence for a hydride bridge in the Ni-R form of [NiFe] hydrogenase has been obtained using iron-specific nuclear resonance vibrational spectroscopy (NRVS). The Ni-H-Fe wag mode at 675 cm(-1) is the first spectroscopic evidence for a bridging hydride in Ni-R as well as the first iron-hydride-related NRVS feature observed for a biological system. Although density function theory (DFT) calculation assisted the determination of the Ni-R structure, it did not predict the Ni-H-Fe wag mode at ∼ 675 cm(-1) before NRVS. Instead, the observed Ni-H-Fe mode provided a critical reference for the DFT calculations. While the overall science about Ni-R is presented and discussed elsewhere, this article focuses on the long and strenuous experimental journey to search for and experimentally identify the Ni-H-Fe wag mode in a Ni-R sample. As a methodology, the results presented here will go beyond Ni-R and hydrogenase research and will also be of interest to other scientists who use synchrotron radiation for measuring dilute samples or weak spectroscopic features.