Zinc is an essential metal for all organisms, as it participates in the structure and/or function of many proteins. However, zinc excess is as deleterious to cells as zinc deficiency. A genome-wide study of the transcriptomic response to high zinc in S. cerevisiae performed in our laboratory allowed the identification of a zinc hyper-tolerant deletion mutant (pif1Δ), which lacks the Pif1 DNA helicase. Further molecular characterization of this strain phenotype revealed that the lack of Pif1 leads to increased iron accumulation, redistribution of the aconitase protein to mitochondria, and also a loss of aconitase activity, despite normal Aco1 protein levels being present, probably due to the epistasis in protecting mtDNA between PIF1 and ACO1. The results presented in this work focus now on the characterization of different features related to the Aco1 protein and activity in yeast and the tolerance to high zinc. Hence, multiple phenotypic traits related to metal metabolism, namely Aco1 protein content and activity levels, succinate dehydrogenase activity, citrate levels, metal content, BPS influence in cultures, and the range of transcription of some iron metabolism related genes, have been analyzed for several strains, some of them constructed to this end, including BY4741, the deletants pif1Δ and aco1Δ, and the aco1 mutants aco1Δ-d4, aco1-C448S, aco1-R476S and aco1-R668S. Overall, lack of Aco1 enzymatic activity in mitochondria, citrate accumulation and lack of activity of [Fe-S] enzymes, e.g. succinate dehydrogenase, appear to be direct molecular indicators of increased zinc tolerance in S. cerevisiae.