F1-ATPase (F1) is a rotary motor protein fuelled with ATP hydrolysis. Although most residues forming the catalytic sites are on the β subunits, the α subunits contain 1 catalytically critical and well-conserved arginine residue, termed the arginine finger, on which the focus of this study is. In this study, we examined the arginine finger catalysis by single molecule measurements for a mutant of F1 in which the arginine finger is substituted with an unnatural amino acid of a lysine analogue, 2,7-diaminoheptanoic acid (Lyk). The use of Lyk, of which the side-chain is elongated by one CH2 unit so that its length to the terminal nitrogen of amine is set to be the same as that of arginine, allowed us to resolve key factors in the arginine finger catalysis, i.e., geometric chain length matching and chemical properties of the terminal groups. Rate measurements by single molecule observations showed that not the geometric chain length matching but the terminal guanidium group in arginine finger is indispensable for catalyzing ATP. By contrast, the geometric matching of the side-chain length prevented the inhibited-state of F1. Therefore, our study clarified the critical factor of the specific residue in detail, so that now we expect that incorporation of unnatural amino acids can widely extend biochemical approaches for elucidation of molecular mechanism of protein functions at a high chemical resolution in the future.