Multiple active site residues are important for photochemical efficiency in the light-activated enzyme protochlorophyllide oxidoreductase (POR).
Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth.
Centre for Synthetic Biology of Fine and Speciality Chemicals, Manchester Institute of Biotechnology, School of Chemistry, The University of Manchester, Manchester, M1 7DN, UK.
Binuraj R K
BR
Menon
Centre for Synthetic Biology of Fine and Speciality Chemicals, Manchester Institute of Biotechnology, School of Chemistry, The University of Manchester, Manchester, M1 7DN, UK.
Samantha J O
SJ
Hardman
Centre for Synthetic Biology of Fine and Speciality Chemicals, Manchester Institute of Biotechnology, School of Chemistry, The University of Manchester, Manchester, M1 7DN, UK. Electronic address: nigel.scrutton@manchester.ac.uk.
Nigel S
NS
Scrutton
Centre for Synthetic Biology of Fine and Speciality Chemicals, Manchester Institute of Biotechnology, School of Chemistry, The University of Manchester, Manchester, M1 7DN, UK. Electronic address: Derren.Heyes@manchester.ac.uk.
Derren J
DJ
Heyes
D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D007700:Kinetics; D008027:Light; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011521:Protochlorophyllide / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013053:Spectrophotometry
D011521:Protochlorophyllide; D009249:NADP; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase
Enzyme catalysis; Light activation; Photochemistry; Protochlorophyllide oxidoreductase (POR); Site-directed mutagenesis