Photochemical and biochemical characterization of GUN4 reactions with protoporphyrin IX and phycocyanobilin

These data include characterization of the reactions of GUN4 protein with bound tetrapyrroles, using GUN4 from the model green alga Chlamydomonas. In almost all oxygenic photosynthetic organisms, GUN4 functions as a fourth \"accessory\" subunit of magnesium chelatase (MgCh), the enzyme carrying out the committed step in chlorophyll biosynthesis. GUN4 is able to bind a wide range of tetrapyrroles, including the protoporphyrin IX (PPIX) substrate and magnesium protoporphyrin IX (MgPPIX) product of the MgCh reaction. GUN4 can also bind linear tetrapyrroles (bilins) such as phycocyanobilin (PCB), and past work has shown that the GUN4•PCB complex stimulates MgCh activity by an order of magnitude in vitro and that both GUN4 and bilin are required for maintenance of the catalytic subunit of MgCh (CHLH1) in vivo. The current dataset examines the behavior of the GUN4/PCB/PPIX system both in vivo and in vitro. In vitro measurements used Chlamydomonas GUN4 protein purified after recombinant expressi..., Tetrapyrrole levels were measured using published procedures, with initial solvent extraction and subsequent separation using high-performance liquid chromatography (HPLC). Protein levels were measured using Western blotting with subsequent quantitation by densitometry. Trp oxidation used tandem LC-MS, and in vivo measurements used a Strep-tagged allele of GUN4 to facilitate isolation from Chlamydomonas cells. Complementation of the gun4 allele by the Strep-tagged allele was confirmed using growth assays with and without acetate under a range of light conditions that included high light stress and exposure to near-UV, both conditions that are associated with growth defects in gun4 cells. Static fluorescence measurements were taken on a QM-6/2005SE fluorimeter at room temperature (Photon Technology International 814 Series).  Fluorescence lifetime measurements were taken using an EasyLife lifetime fluorescence spectrometer (PTI) with a 280 nm LED source, using a 320 nm long-pass Sch..., # Photochemical and biochemical characterization of GUN4 reactions with protoporphyrin IX and phycocyanobilin

Dataset DOI: [10.5061/dryad.47d7wm3vq](https://doi.org/10.5061/dryad.47d7wm3vq)

## Description of the data and file structure

Four sub-directories span a range of data types, all presented as tab-delimited text files. Detailed information about the measurements and reported values are included in a README text file in each directory. The four sub-directories are as follows:

1.     in_vivo_tetrapyrrole_protein_levels

This sub-directory includes in vivo measurements of tetrapyrrole metabolites and protein levels in *Chlamydomonas* cells.

PPIX and MgPPIX levels are reported for wild-type cells (using the 4A+ background), gun4 cells, hmox1 cells, and gun4 hmox1 cells. The different mutations are loss-of-function for GUN4 protein, for the plastid heme oxygenate HMOX1 required for PCB biosynthesis in *Chlamydomonas*, or for both.

Protein levels were measured as a function of ti..., ,