Protein-Induced Excited-State Dynamics of Protochlorophyllide

Abstract

The light-driven NADPH:protochlorophyllide oxidoreductase (POR) is a key enzyme of chlorophyll biosynthesis in angiosperms. POR's unique requirement for light to become catalytically active makes the enzyme an attractive model to study the dynamics of enzymatic reactions in real time. Here, we use picosecond time-resolved fluorescence and femtosecond pump-probe spectroscopy to examine the influence of the protein environment on the excited-state dynamics of the substrate, protochlorophyllide (PChlide), in the enzyme/substrate (PChlide/POR) and pseudoternary complex including the nucleotide cofactor NADP(+) (PChlide/NADP(+)/ POR). In comparison with the excited-state processes of unbound PChlide, the lifetime of the thermally equilibrated S(1) excited state is lengthened from 3.4 to 4.4 and 5.4 ns in the PChlide/POR and PChlide/NADP(+)/POR complex, whereas the nonradiative rates are decreased by ∼30 and 40%, respectively. This effect is most likely due to the reduced probability of nonradiative decay into the triplet excited state, thus keeping the risk of photosensitized side reactions in the enzyme low. Further, the initial reaction path involves the formation of an intramolecular charge-transfer state (S(ICT)) as an intermediate product. From a strong blue shift in the excited-state absorption, it is concluded that the S(ICT) state is stabilized by local interactions with specific protein sites in the catalytic pocket. The possible relevance of this result for the catalytic reaction in the enzyme POR is discussed.

Excited‐State Charge Separation in the Photochemical Mechanism of the Light‐Driven Enzyme Protochlorophyllide Oxidoreductase

“…[11,12] Similar findings have also been reported for apseudo-ternary (i.e.non-productive) POR-Pchlide-NADP + complex where there was astrong blue-shift in the ESA bands associated with the ICT state. [19] Hence,i ti sp ossible that hydrogen-bonding effects between residues in the active site of the enzyme and the Pchlide molecule may considerably change or stabilize the ICT state.T he negative feature at approximately 675 nm in the POR-Pchlide-NADPH is ar esult of the Chlide product that accumulates during the course of the measurements. [20] To ensure that this signal did not mask the actual excited-state spectral changes in the POR reaction cycle it was minimized by limiting the length of data acquisition to less than 5minutes.…”

“…This is in agreement with previous ultrafast time-resolved IR measurements,w hich suggested that this step is associated with structural changes to the C13 carbonyl and delocalized C=Cand C=Nmodes. [19] This is …”

“…[3] An umber of time-resolved transient spectroscopy studies have identified different short-lived Pchlide* species,both in the isolated pigment [11][12][13][14][15] and in the ternary enzyme-substrate complex. [16][17][18][19][20] TheP chlide molecule is intrinsically very reactive with multi-exponential excited-state dynamics [11][12][13][14][15][16] and the formation of an umber of Pchlide excited-state species within afew nanoseconds,including an intramolecular charge-transfer (ICT) complex [11][12][13][14][15] and atriplet state. [13,15] A detailed understanding of the initial ultrafast steps in the ternary enzyme-substrate (POR-Pchlide-NADPH) complex that lead directly to the chemistry of Pchlide photoreduction has proved more challenging.…”

“…[13,15] A detailed understanding of the initial ultrafast steps in the ternary enzyme-substrate (POR-Pchlide-NADPH) complex that lead directly to the chemistry of Pchlide photoreduction has proved more challenging. [16][17][18][19][20] Recent studies have shown [20] Moreover, conventional ultrafast pump-probe spectroscopy measurements are typically limited to af ew nanoseconds,w hich has prevented the identification of any long-lived excited-state intermediates prior to the hydride and proton transfer chemistry.T oo vercome this problem we have now used time-resolved UV/Vis and IR spectroscopy techniques that provide complete temporal resolution over the picosecondmicrosecond time range.Consequently,this has allowed us to identify all of the excited-state intermediates in the catalytic cycle of POR and to propose amechanism for harnessing the light energy to drive the subsequent hydride and proton transfer steps on the microsecond timescale.…”

Cell growth defect factor 1 is crucial for the plastid import of NADPH:protochlorophyllide oxidoreductase A in Arabidopsis thaliana

“…6). Mutagenesis and homology modeling studies have provided important insights into the catalytic mechanism of POR (7)(8)(9)(10)(11). All three POR isoforms are nuclear gene products that must be imported posttranslationally into the plastids (12,13).…”