Alkane monooxygenase (AlkB) is the dominant enzyme that catalyzes the oxidation of liquid alkanes in the environment. Two recent structural models derived from cryo-electron microscopy (cryo-EM) reveal an unusual active site: a histidine-rich center that binds two iron ions without a bridging ligand. To ensure that potential photoreduction and radiation damage are not responsible for the absence of a bridging ligand in the cryo-EM structures, spectroscopic methods are needed. We present the results of extended X-ray absorption fine structure (EXAFS) experiments collected under conditions where photodamage was avoided. Careful data analysis reveals an active site structure consistent with the cryo-EM structures in which the two iron ions are ligated by nine histidines and separated by at least 5 Å. The EXAFS data were used to inform structural models for molecular dynamics (MD) simulations. The MD simulations corroborate EXAFS observations that neither of the two conserved carboxylate-containing residues (E281 and D190) near the active site are likely candidates for metal ion bridging. Mutagenesis experiments, spectroscopy, and additional MD simulations were used to further explore the role of these carboxylate residues. A variant in which a carboxylate containing residue (E281) was changed to a methyl residue (E281A) showed little change in pre-edge features, consistent with the observation that it is not essential for activity and hence unlikely to serve as a bridging ligand at any point in the catalytic cycle. D190 variants had substantially diminished activity, suggesting an important role in catalysis not yet fully understood.