Novel copper-regulated, candidate diiron enzymes with a role in plastid iron metabolism

Abstract

Chlamydomonas reinhardtii, a chloroplast-containing eukaryote amenable to genetic and molecular manipulation, is a suitable model for the study of copper metabolism in photosynthetic organisms. Copper enzymes function in fundamental metabolic pathways like photosynthesis (plastocyanin), respiration (cytochrome oxidase) and iron assimilation (membrane multicopper oxidase). Chlamydomonas survives copper deficiency by activating adaptive mechanisms, which include ¿ regulated degradation of the major copper protein, plastocyanin, so that copper can be re-distributed to other enzymes; transcriptional activation of Cyc6 encoding a cytochrome that substitutes for plastocyanin in vivo; and activation of a copper assimilatory pathway involving a reductase and a transporter. A genetically-defined master regulator, Crr1, is required for these adaptive responses. Recently, we have identified two candidate diiron enzymes, Crd1 and Cth1, associated with thylakoid membranes. The proteins are 66% identical but display distinct patterns of expression; Crd1 is expressed primarily in ¿Cu cells while Cth1 is expressed in +Cu cells. The absence of Crd1 in copper-deficient cells results in a chlorotic phenotype reminiscent of iron-deficiency; yet crd1 mutants are not iron-deficient under these conditions. One model that accounts for the incongruity is that cells lacking Crd1 suffer only a localized iron-deficiency ¿ as if the enzyme functioned in iron-delivery to photosystem I. Mis-expression of Cth1 can almost completely rescue the crd1 phenotype in ¿Cu cells suggesting that they are functionally very similar. One model is that the two enzymes may have different physical or kinetic properties so that the cell can handle plastid iron metabolism in response to variation in copper enzyme function.