Rhodopsins are heptahelical transmembrane photoactive protein families: type 1 (microbial rhodopsins) and type 2 (animal rhodopsins). Both families share similar topologies and chromophore retinal, which is linked covalently as a protonated Schiff base to a Lys at the transmembrane 7 helix. Recently, through functional metagenomics analysis, we reported an unnoticed diverse family, heliorhodopsins (HeRs), which are abundant and distributed globally in archaea, bacteria, eukarya, and viruses. The sequence identity is <15% between HeRs and type 1 rhodopsins, so that many aspects of the molecular properties of HeRs remain unknown. Herein, to gain information about the residues responsible for the interaction with the chromophore, we applied Ala scanning to 30 candidate residues in HeR 48C12. As a result, 12 mutants showed no absorption change, eight exhibited a spectral blue-shift, six exhibited a spectral red-shift, and four did not form a pigment. R104, Y108, G145, and K241 play crucial roles in pigment formation. A combination of single mutants successfully engineered pigments absorbing at 523 nm (S112A/M141A) and 571 nm (H80A/S237A), covering more than ∼50 nm. These results provide fundamental knowledge about the molecular properties of HeRs.