OPEN Dark-operative protochlorophyllide

SUBJECT AREAS: oxidoreductase generates substrate ENZYME MECHANISMS BIOENERGETICS radicals by an iron-sulphur cluster in

Received biosynthesis 5 December 2013 Jiro Nomata1*, Toru Kondo2{, Tadashi Mizoguchi3, Hitoshi Tamiaki3, Shigeru Itoh2{ & Yuichi Fujita1 Accepted 9 June 2014 1Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan, 2Graduate Published School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan, 3Graduate School of Life Sciences, 26 June 2014 Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan.

Correspondence and Photosynthesis converts solar energy to chemical energy using (Chls). In a late stage of biosynthesis of Chls, dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR), a requests for materials -like enzyme, reduces the C175C18 double bond of Pchlide and drastically changes the spectral should be addressed to properties suitable for photosynthesis forming the parental ring for Chl a. We previously proposed Y.F. (fujita@agr. that the spatial arrangement of the proton donors determines the stereospecificity of the Pchlide reduction nagoya-u.ac.jp) based on the recently resolved structure of the DPOR catalytic component, NB-protein. However, it was not clear how the two-electron and two-proton transfer events are coordinated in the reaction. In this study, we demonstrate that DPOR initiates a single electron transfer reaction from a [4Fe-4S]-cluster (NB-cluster) to * Current address: Pchlide, generating Pchlide anion radicals followed by a single proton transfer, and then, further electron/ Chemical Resources proton transfer steps transform the anion radicals into (Chlide). Thus, DPOR is a unique Laboratory, Tokyo iron-sulphur enzyme to form substrate radicals followed by sequential proton- and electron-transfer steps with the protein folding very similar to that of nitrogenase. This novel radical-mediated reaction supports Institute of Technology, the biosynthesis of Chl in a wide variety of photosynthetic organisms. Nagatsuta, 4259, Midori-ku, Yokohama, 226-8503. n Chl biosynthesis, Pchlide reduction is the final reaction forming the conjugated p-system, the chlorin structure of Chl a, by the stereo-specific reduction of the C175C18 double bond (Fig. 1a). There are two { Current address: evolutionary unrelated Pchlide reductases, one is the light-dependent Pchlide oxidoreductase (LPOR)1, which I 2 Department of Physics, requires light for catalysis, and the other is DPOR , which operates in a light-independent manner. Tokyo Institute of LPOR is composed of a single polypeptide that belongs to short-chain dehydrogenase/reductase (SDR) family. Technology, LPOR is the responsible enzyme for light-dependent greening of angiosperms because LPOR operates as the sole Pchlide reductase in angiosperms1. Since the LPOR reaction is triggered by light, this enzyme provides a unique O-okayama, Meguro-ku, opportunity to study the reactions by ultrafast timescale. Although crystal structure of LPOR has not yet been Tokyo 152-8551, revealed, several spectroscopic reaction intermediates were detected by triggering the reaction with a short laser Japan. pulse allowing to elucidate the catalytic cycle of LPOR3,4. These kinetic analyses suggested that a hydride transfer from NADPH to C18 is induced by light activation of Pchlide followed by several light-independent reactions {Current address: including a proton transfer from a Tyr residue to C17, release of Chlide and exchange of cofactors5. Centre for Gene In oxygenic photosynthetic organisms except for angiosperms, DPOR serves as the determinant enzyme Research, Nagoya mediating the greening in the dark. Anoxygenic photosynthetic