In preparing the histidine Nε2–heme vinyl Cα crosslink, care must be exercised in the choice of a reducing agent to avoid heme and protein damage, as occasionally caused by the oxidative by-products of aerobic DT reduction. Anaerobic DT treatment or reduction with alternative agents such as a ferredoxin system [56] if sufficiently powerful may be preferable. (3) It is possible to engineer the crosslink in a b heme protein using relatively mild reaction conditions. Although detailed kinetic analysis was not performed on CtrHb variants, the products are consistent with the mechanism described previously [21], in which case the pKa of the reactive histidine, in addition to its ability to adopt the geometry of the product, is an important factor