Hydroxamic acids, known as iron chelators, have recently been widely used as a key functional group of potential therapeutics targeting at zinc proteinases such as matrix metalloproteinases involved in cancers and at other drug targets associated with cardiovascular diseases, AIDS, and Alzheimer's disease. However, the protonation states of zinc-coordinated hydroxamic acids in proteins and relative affinities of hydroxamic acids for Zn2+ are still unclear due to the intricacy of the hydroxamic acid structures. Here, we report a comprehensive ab initio study of stable configurations and tautomers of neutral and deprotonated, Zn2+-coordinated acetohydroxamic acid and its N-methyl analogue in the gas-phase employing the B3LYP/6-311+G(2d,2p) method. The results suggest that both zinc-coordinated acetohydroxamic and N-methylacetohydroxamic acids exist in the oxygen-deprotonated Z-keto form with their two oxygen atoms coordinating to zinc in proteins in which the acidic amino acid side chains serve as a proton acceptor. This conclusion is consistent with a survey of experimentally determined protein 3D structures complexed with zinc-coordinated hydroxamic acids documented in the Protein Data Bank. The results also suggest that the zinc affinity of N-methylacetohydroxamic acid is 11 kcal/mol higher than that of acetohydroxamic acid and is up to 43 kcal/mol higher than those of common zinc ligands in proteins. It thus cautions the use of N-methylacetohydroxamic acid as a functional group in rational design of inhibitors for zinc proteinases, since it may interact with other zinc proteins due to its high affinity for zinc.