Monofunctional heme-catalases have been studied for many decades but there is still an incomplete understanding of why such a large tetrameric protein with deeply buried active sites is required to accomplish such a simple reaction as H2O2 dismutation. Catalase accomplishes this reaction at a high rate although water at 55 M is expected to compete with H2O2 for the enzyme's active site. Using molecular dynamics simulations we addressed the question as to how catalase selects H2O2 in water. Selection is accomplished through different mechanisms: higher residence time of H2O2 in the vicinity of certain prevalent amino acid residues at the protein surface and substrate channel, coordinated motion of the main passage amino acids that is increased in the presence of H2O2, a gate valve mechanism consisting of the motion of two contiguous phenylalanine residues that drive water molecules out of the final section of the substrate channel, a hydrophobic barrier before the active site that was crossed more easily by H2O2 which kept most of its hydrogen bonds while passing, and finally an increased residence time for H2O2 at the active site. These mechanisms, based on the physicochemical differences between H2O2 and water, provide an explanation as to why such a large tetrameric protein with deeply buried active sites is required to accomplish efficient H2O2 dismutation. Proteins 2014; 82:45–56. © 2013 Wiley Periodicals, Inc.