Cysteinate protonation and water hydrogen bonding at the active-site of a nickel superoxide dismutase metallopeptide-based mimic: implications for the mechanism of superoxide reduction.

Abstract

Nickel-containing superoxide dismutase (NiSOD) is a mononuclear cysteinate-ligated nickel metalloenzyme that catalyzes the disproportionation of superoxide into dioxygen and hydrogen peroxide by cycling between Ni(II) and Ni(III) oxidation states. All of the ligating residues to nickel are found within the first six residues from the N-terminus, which has prompted several research groups to generate NiSOD metallopeptide-based mimics derived from the first several residues of the NiSOD sequence. To assess the viability of using these metallopeptide-based mimics (NiSOD maquettes) to probe the mechanism of SOD catalysis facilitated by NiSOD, we computationally explored the initial step of the O2(-) reduction mechanism catalyzed by the NiSOD maquette {Ni(II)(SOD(m1))} (SOD(m1) = HCDLP CGVYD PA). Herein we use spectroscopic (S K-edge X-ray absorption spectroscopy, electronic absorption spectroscopy, and circular dichroism spectroscopy) and computational techniques to derive the detailed active-site structure of {Ni(II)(SOD(m1))}. These studies suggest that the {Ni(II)(SOD(m1))} active-site possesses a Ni(II)-S(H(+))-Cys(6) moiety and at least one associated water molecule contained in a hydrogen-bonding interaction to the coordinated Cys(2) and Cys(6) sulfur atoms. A computationally derived mechanism for O2(-) reduction using the formulated active-site structure of {Ni(II)(SOD(m1))} suggests that O2(-) reduction takes place through an apparent initial outersphere hydrogen atom transfer (HAT) from the Ni(II)-S(H(+))-Cys(6) moiety to the O2(-) molecule. It is proposed that the water molecule aids in driving the reaction forward by lowering the Ni(II)-S(H(+))-Cys(6) pK(a). Such a mechanism is not possible in NiSOD itself for structural reasons. These results therefore strongly suggest that maquettes derived from the primary sequence of NiSOD are mechanistically distinct from NiSOD itself despite the similarities in the structure and physical properties of the metalloenzyme vs the NiSOD metallopeptide-based models.