Molecular dynamics simulations of the photoactive protein nitrile hydratase.

Nitrile hydratase (NHase) is an enzyme used in the industrial biotechnological production of acrylamide. The active site, which contains nonheme iron or noncorrin cobalt, is buried in the protein core at the interface of two domains, alpha and beta. Hydrogen bonds between betaArg-56 and alphaCys-114 sulfenic acid (alphaCEA114) are important to maintain the enzymatic activity. The enzyme may be inactivated by endogenous nitric oxide (NO) and activated by absorption of photons of wavelength lambda < 630 nm. To explain the photosensitivity and to propose structural determinants of catalytic activity, differences in the dynamics of light-active and dark-inactive forms of NHase were investigated using molecular dynamics (MD) modeling. To this end, a new set of force field parameters for nonstandard NHase active sites have been developed. The dynamics of the photodissociated NO ligand in the enzyme channel was analyzed using the locally enhanced sampling method, as implemented in the MOIL MD package. A series of 1 ns trajectories of NHases shows that the protonation state of the active site affects the dynamics of the catalytic water and NO ligand close to the metal center. MD simulations support the catalytic mechanism in which a water molecule bound to the metal ion directly attacks the nitrile carbon.