Biological systems activate O2 using many mechanisms, but in nearly all cases, the activation process is regulated to assure specificity. The nature of these regulatory aspects of the reaction must be understood before the true nature of the underlying chemistry can be described with certainty. Most metal-containing oxygenases utilize amino acids in the second sphere and beyond to regulate the O2 activation reaction. One example of this is seen in the mechanism of substrate selectivity by methane monooxygenase. The regulatory protein MMOB binds to the active site-containing MMOH and appears to create a pore sized for methane into the active site. This controls access and therefore the preferred substrate. Also, the complex appears to cause quantum tunneling to dominate in C–H bond cleavage reaction for methane, selectively increasing the rate for this substrate. Both effects can be altered by mutagenesis of MMOB, potentially broadening the substrate range of the enzyme. Second sphere effects are also important in determining the position of ring cleavage for catecholic ring cleaving dioxygenases. Intermediates throughout the catalytic cycle of homoprotocatechuate 2,3-dioxygenase can be detected by using the chromophoric substrate 4-nitrocatechol (4NC). Upon mutation of the second sphere residue histidine 200 to asparagine (H200N), the rate of reaction of the Fe-oxy intermediate is greatly slowed, allowing its detection for the first time when using either 4NC or the natural substrate 3,4-dihydroxyphenylacetate (HPCA). HPCA cleavage occurs in the usual proximal extradiol position by this mutant, but 4NC is oxidized to the quinone without ring cleavage. Use of the alternative substrate 2,3-dihydroxybenzoate results in distal extradiol cleavage for the wild type enzyme, but intradiol cleavage for the H200-phenylalanine mutant. Thus, control of the second sphere allows the enzyme to design a specific catalyst that gives only one of the four potential types of products. This insight can be used to design specific enzyme oxidation catalysts.