Proteins of the Rieske and Rieske-type family contain a [2Fe-2S] cluster with mixed ligation by two histidines and two cysteines, and play important roles in various biological electron transfer reactions. We report here the comparative orientation-selected ESEEM and HYSCORE studies of the reduced clusters from two hyperthermophilic Rieske-type proteins; a high-potential, archaeal Rieske protein called sulredoxin (SDX) from Sulfolobus tokodaii with weak homology to the cytochrome bc-associated Rieske proteins, and a low-potential, archaeal homolog of an oxygenase-associated Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus. (14)N ESEEM and HYSCORE spectra of SDX and ARF show well-defined variations, which are primarily determined by changes of quadrupole couplings (up to 50% depending on the selected orientation) of the two coordinated nitrogens. These are due to variations in coordination geometry of the histidine imidazole ligands rather than to variations of hyperfine couplings of these nitrogens, which do not exceed 8-10%. The measured quadrupole couplings and their differences in the two proteins are consistent with those calculated using the reported crystal structures of high- and low-potential Rieske proteins. These results suggest that exploration of quadrupole tensors might provide a more accurate method for characterization of the histidine coordination in different proteins and mutants than hyperfine tensors, and might have potential applications in a wider range of biological systems.