BM3h-8C8 Mutant of the heme domain of the bacterial cytochrome P450-BM3 ()

BM3h-8C8 is a mutant of the heme domain of the bacterial cytochrome P450-BM3 (BM3h) from , and it was generated by Shapiro et al. with directed evolution of BM3h for magnetic resonance imaging (MRI) of dopamine (1). Dopamine is a catecholamine neurotransmitter produced mainly by the medulla of the adrenal glands and the nervous tissues, including the substantia nigra and the ventral tegmental area (2, 3). In neurons, synthetic dopamine is packaged into vesicles and then released into the synapse in response to presynaptic action potentials. Dopamine also acts as a neurohormone, inhibiting prolactin release from the pituitary gland (4). Dopamine is of further significance because of its roles in learning, reward, and motor coordination, and because of the dysfunction of dopaminergic systems underlying addiction and neurodegenerative diseases (5-8). Radionuclides have been investigated for imaging dopamine and its receptors , but both positron emission tomography and single-photon emission tomography have low spatial and temporal resolution in spite of high sensitivity (9-12). Shapiro et al. developed a dopamine sensor for MRI using directed evolution of BM3h (1). BM3h, a 53-kDa moiety, contains a single iron(III) atom bound to a hemin prosthetic group (13). The iron ion is the site of oxygen binding; when oxygen is not bound, a water molecule fills the site with a weak bond. Interaction of the heme iron with exchanging water molecules at this axial site could promote relaxation and thus modulate MRI contrast (1). Studies by Shapiro et al. revealed an value ( relaxivity; 1/1) of 1.23 ± 0.07 mM−s− for BM3h in phosphate-buffered saline without ligands. Addition of a saturating quantity of arachidonic acid, a natural BM3h substrate, resulted in an value of 0.42 ± 0.05 mM−s−. This ligand-induced decrease in relaxivity, arising from the displacement of water molecules at the BM3h heme, enabled quantitative sensing of arachidonic acid with MRI. Further studies by Shapiro et al. showed that addition of 1 mM dopamine to BM3h induced a decrease in the value to 0.76 ± 0.03 mM−s−, indicating that dopamine directly replaces water as an axial metal ligand in the BM3h substrate-binding pocket. The apparent dissociation constant () for arachidonic acid was 6.8 ± 0.5 μM, and the for dopamine was 990 ± 110 μM. These results suggest that BM3h could serve as a platform for molecular sensor engineering (1, 14, 15). Using the platform of BM3h, Shapiro et al. generated a series of MRI sensors for dopamine by decreasing the affinity for arachidonic acid, increasing the affinity for dopamine, and enhancing the relaxivity changes upon ligand binding with the directed evolution technique (1). Directed evolution is a molecular engineering method that utilizes successive rounds of mutagenesis and selection to generate proteins with novel functionality, starting from a molecule with some of the desired properties of the end product (14, 15). BM3h-8C8 is one of the BM3h-based mutants with the best combination of relaxivity change, improved affinity for dopamine, and decreased affinity for arachidonic acid (1).