Human mitochondrial RNase P protein 1 (MRPP1) and 2 (MRPP2) form a methyltransferase complex with N1-methylation activity towards purine-9 on mitochondrial tRNA. A third component, mitochondrial RNase P protein 3 (MRPP3), adds RNase P 5'-end ribonuclease processing activity to the complex. MRPP2 alone is a dehydrogenase reductase (SDR) involved in many reactions e.g. steroid metabolism and isoleucine β-oxidation. Inherited MRPP2 missense mutations cause the rare neurological HSD10 disease, where, due to the multifunctional nature of MRPP2, no clear genotype-phenotype relationship has been established. In this study, investigations are performed on the molecular properties of the individual proteins, as well as interactions within the methyltransferase/RNase P MRPP1-MRPP2-MRPP3 complex, to shed light on disease-causing mutations. At the molecular level, the N-terminal region of MRPP1, with no known structural fold, is responsible for dimerisation, whilst the C-terminal region contains a methyltransferase (MT) domain. The structure of the MRPP1 MT-domain to 1.96 Å resolution reveals the binding of the methyl donor S-adenosyl-methionine (SAM). Assembly of the MRPP1-MRPP2 methyltransferase complex requires intact proteins, and formation of the MRPP1-MRPP2-MRPP3 RNase P complex further requires the pre-tRNA substrate. Low-resolution small angle X-ray scattering (SAXS) models of the complexes, with and without tRNA substrate, provide clues into the protein-tRNA interaction. Reported patient mutations in MRPP2 alters the biochemical properties of the protein with each effect correlating with the structural location of the mutation. From the biochemical data, a correlation exists between phenotype and MRPP2 protein abundance. A novel missense mutation, causing a mild phenotype, show devastating effects on all functions involving MRPP2, thus no clear correlation exists between disease severity and residual activity. A patient mutation in MRPP3, leading to the rare disorder Perrault syndrome, targets the active site and leads to a decrease in product formation. The mutation does not affect MRPP1-MRPP2-MRPP3 complex assembly, hence agreeing well with it being a catalytic mutant. Together, these structural and biochemical data provide a better understanding of the molecular facets affected by HSD10 disease patient mutations, to further explore the diverse disease severity observed in patients. Long term, this will hopefully lead to novel treatments designed for patients suffering from HSD10 disease.