Probing reactive sites within the Photosystem II manganese cluster: Evidence for separate populations of manganese that differ in redox potential

Abstract

When the oxygen evolving complex of Photosystem II is depleted of essential cofactor atoms (Ca²⁺ and Cl⁻) by a high ionic strength treatment that extracts 23 and 17 kDa extrinsic polypeptides, Mn²⁺ can be released by several large reductants (hydroquinone (H₂Q), N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), methyl derivatives of hydroxylamine). This reactivity can be slowed if the enzyme is reconstituted with Ca²⁺. For TMPD, data from EPR and X-ray absorption spectroscopy indicate that Ca²⁺ reconstitution restricts initial Mn reduction to a site which contains a Mn⁴⁺ atom. Dimethylhydroxylamine (DMHA), a much smaller Mn reductant, is unable to reduce the cluster under these same conditions, even though DMHA and TMPD can generate Mn²⁺ from the Mn cluster in the absence of Ca²⁺. These reductants differ in redox potential by about 300 mV, and it is likely that the higher potential reductant, DMHA (+550 mV), is restricted to initially reacting with a Mn³⁺ species that is screened by the Ca²⁺ atom and is incapable, in the presence of Ca²⁺, of reducing lower potential atoms of the cluster that do react with TMPD. Reduction of the reactive Mn³⁺ species by DMHA in the absence of Ca²⁺, however, subsequently allows reduction of the remaining 3 Mn. Such an arrangement of metals and potentials in the cluster is in accord with models for the site of water oxidation and for the structure of the Ca–Mn₄ cluster.