Methylthiotransferase that catalyzes the conversion of N6-(dimethylallyl)adenosine (i(6)A) to 2-methylthio-N6-(dimethylallyl)adenosine (ms(2)i(6)A) at position 37 (adjacent to the 3'-end of the anticodon) of four mitochondrial DNA-encoded tRNAs (Ser(UCN), Phe, Tyr and Trp) (PubMed:25738458). Essential for efficient and highly accurate protein translation by the ribosome (PubMed:25738458). Specifically inhibits CDK5 activation by CDK5R1 (By similarity). Essential for efficient mitochondrial protein synthesis and respiratory chain (PubMed:25738458). [sulfur carrier]-SH + AH2 + N(6)-dimethylallyladenosine(37) in tRNA + 2 S-adenosyl-L-methionine = 2-methylsulfanyl-N(6)-dimethylallyladenosine(37) in tRNA + 5'-deoxyadenosine + [sulfur carrier]-H + A + 2 H(+) + L-methionine + S-adenosyl-L-homocysteine Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. Interacts with CDK5R1 (p35 form). CDK5RAP1, CDK5RAP2 and CDK5RAP3 show competitive binding to CDK5R1. Probably forms a complex with CDK5R1 and CDK5. Expressed in brain, liver, skeletal muscle and heart. Knockout mice show a deficiency in ms(2)i(6)A modification, resulting in impaired mitochondrial protein synthesis, which leads to respiratory defects. Belongs to the methylthiotransferase family. MiaB subfamily.