Interactions between diphenylcarbazide, zinc, cobalt, and manganese on the oxidizing side of photosystem II.

The inhibition of DPC-mediated DCIP photoreduction by exogenous MnCl2 in Tris-treated photosystem II (PSII) membrane fragments has been used to probe for amino acids on the PSII reaction center proteins, including D1His337, that provide ligands for binding manganese [Preston, C., & Seibert, M. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) Vol. I, pp 925-928, Kluwer Academic Publishers, Dordrecht, The Netherlands; Preston, C., & Seibert, M. (1991) Biochemistry 30, 9615-9624 and 9625-9633]. At a concentration of 200 microM, DPC is photooxidized at both a high-affinity and a low-affinity site in PSII at approximately the same initial rate. Addition of 10 microM MnCl2 noncompetitively inhibits DPC photooxidation at the high-affinity site, with a Ki of 1.5 microM, causing a decrease of about 50% in the overall DCIP photoreduction rate. The high-affinity site for Mn binding was deconvoluted into four independent components. In earlier work, the inhibition was attributed to the tight association of either Mn2+ or Mn3+ with the PSII membrane. We report here that inhibition of DPC photooxidation may involve two different types of high-affinity, Mn-binding components: (a) one that is specific for Mn, and (b) others that bind Mn, but may also bind additional divalent cations, such as Zn and Co, that are not photooxidized by PSII. These conclusions are based on the observations that (a) DPC photooxidation can be inhibited by Zn2+ and Co2+; (b) Zn2+ and Co2+ interact with Mn2+ in a nonmutually exclusive manner, suggesting that they may share some binding components with Mn2+; (c) high-affinity Mn2+ (but not Zn2+ or Co2+) inhibition of DPC photooxidation is accompanied by nondecaying fluorescence emission, following a single saturating flash, indicating efficient electron donation by Mn2+ to YZ+; (d) Mn2+ photooxidation in the presence of DPC is not inhibited by Zn2+ or Co2+; and (e) kinetic modeling of the interaction between high-affinity Mn2+ and DPC in PSII indicates inhibition of steady-state Mn2+ photooxidation by DPC, but allows for a single photooxidation of Mn2+. We conclude that Mn inhibition of DPC photooxidation can be used to identify Mn-binding sites of physiological importance, and suggest that the Mn-specific component of the high-affinity, Mn-binding site involves the ligand to the first Mn bound during photoactivation (i.e., Asp170 on D1, as found by other investigators).