Laser Photoexcitation Measurements

Absorption transients at the stated wavelength were measured using laser photoexcitation of dark-assembled enzyme-NADPH-Pchlide ternary complexes. Samples were excited at 450 nm using an optical parametric oscillator of a Q-switched Nd:YAG laser (Brilliant B, Quantel) in a cuvette of 1-cm path length as described previously (12). Rate constants were measured at the stated temperature from the average of at least five time-dependent absorption measurements by fitting to a single exponential function. The formation of an intermediate with an absorbance band at 696 nm represents an initial hydride transfer reaction from NADPH, and the subsequent slower exponential decay in absorbance at 696 nm represents the proton transfer reaction (12). Slower kinetically resolved absorbance changes at 670 nm on the millisecond time scale represent a series of ordered product release and cofactor rebinding events (18). For studies of the temperature dependence of rate constants, data were fitted to the Eyring equation. Buffer conditions for cryogenic studies of hydride and proton transfer were as described previously (12). For viscosity studies, glycerol solutions were prepared by weight, and calculation of solution viscosity was as described (9). The effect of viscosity on the observed rate was fitted to Equation 1 (9, 21) to describe the contribution of the protein friction to the total friction of the system,

where σ, in units of viscosity, is the contribution of the protein friction, and η is the absolute viscosity.

Highly Conserved Localized Dynamics Control Hydride Transfer

To understand how these structural differences have influenced motions coupled to the hydride and proton transfers, we studied the catalytic mechanism and associated protein dynamics of different PORs from a variety of organisms. These included PORs from thermophilic (T. elongatus) and mesophilic (Synechocystis sp. PCC6803) cyanobacteria, a plant POR (A. thaliana POR B), and the basal cyanobacterial species (G. violaceus), which has features that are common to both cyanobacterial and plant PORs (supplemental Fig. S2). The time-dependent spectral changes following laser photoactivation confirmed that the kinetic mechanism of hydride and proton transfer is sequential (i.e. hydride precedes proton transfer) in each enzyme (supplemental Fig. S3). Dynamic information was obtained from an analysis of the temperature dependence of the kinetic isotope effects associated with hydride and proton transfer (4, 6, 8, 12, 25,–29). Also, the solvent viscosity dependence of reaction rates provides further information on the nature of motions linked to the reaction chemistry (9, 21, 30,–32). We used these approaches to interrogate the nature of the motions coupled to hydride and proton transfer in each of the identified POR enzymes.

Rate constants for the initial light-activated hydride transfers are similar for each of the POR enzymes using either NADPH or NADP²H (Table 1). Eyring plots of the hydride transfer step (Fig. 3) indicate an almost identical temperature dependence (range of −70 °C to +50 °C) with a breakpoint at −27 °C for each enzyme. At the higher temperatures, the temperature-dependent kinetic isotope effect (KIE) (Table 1 and supplemental Table S1) confirmed that hydride tunneling is coupled to a dominant promoting motion along the hydride transfer coordinate (12, 25,–29). Conversely, below −27 °C, the KIE was essentially independent of temperature (Table 1 and supplemental Table S1), indicating that promoting motions are “frozen” at these temperatures with a consequent higher energetic cost to hydride tunneling, as reflected in the increased enthalpy of the reaction (12). Remarkably, for the hydride tunneling step, the dynamic profile of each POR enzyme was identical to that we reported previously for hydride tunneling in T. elongatus POR (12), regardless of the evolutionary lineage of the enzyme. The lack of a solvent viscosity dependence on the hydride transfer reaction for all PORs studied herein (see Fig. 5) points to the importance of localized (and not extended) dynamics in the hydride transfer reaction, as we previously inferred for T. elongatus POR (9). The NADPH-binding site is highly conserved in all PORs (Fig. 2A), and single residue changes in this region of the enzyme are known to compromise hydride transfer by substantially affecting the lifetime of the photoexcited state (15, 33, 34). This is consistent with the geometry and active site dynamics being optimally configured in all POR enzymes to enable efficient photochemistry. Evolution has therefore discovered a unique light-dependent solution to overcome a highly unfavorable (2.18 eV) (12) hydride transfer reaction, which will depend on the precise positioning of NADPH and Pchlide in the active site. This stringent geometrical and dynamic solution enables the “difficult” light-driven chemistry in all POR enzymes. The reaction chemistry is controlled by localized dynamics in all PORs through conservation of protein structure in the enzyme active site. The stringent structural requirements of the hydride transfer chemistry are consistent with the known sensitivity of this reaction to minor active site structural perturbations (15, 33, 34).

TABLE 1

Rate constants, isotope effects, and temperature dependences (ΔΔH^‡) of the hydride and proton transfer steps for the various POR enzymes

All rates were measured at 25 °C as described under “Experimental Procedures.”

	T. elongatus	Synechocystis sp. PCC6803	G. violaceus	Arabidopsis POR B
Hydride transfer
KH− (s−1 × 106)	1.87 ± 0.17	1.86 ± 0.18	1.86 ± 0.12	1.81 ± 0.24
KIE	1.91 ± 0.17	1.90 ± 0.16	1.94 ± 0.13	1.91 ± 0.23
ΔΔH‡ (kJ mol−1)a	8.2 ± 0.6	7.0 ± 0.3	6.9 ± 0.7	7.6 ± 0.9
ΔΔH‡ (kJ mol−1)b	(0.4 ± 1.0)	(0.4 ± 1.5)	(0.5 ± 1.2)	(0.3 ± 1.2)
Proton transfer
kH+ (s−1 × 103)	27.4 ± 1.0	30.4 ± 0.6	152.0 ± 5.7	123.3 ± 3.7
SIE	2.01 ± 0.14	1.80 ± 0.08	1.54 ± 0.07	1.50 ± 0.07
ΔΔH‡ (kJ mol−1)	15.6 ± 3.1	17.6 ± 3.7	2.6 ± 2.4	1.0 ± 1.9

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^a These values of ΔΔH^‡ were calculated from the temperature dependence of the KIE measured above the observed breakpoint (−27 °C) (Fig. 3).

^b These values of ΔΔH^‡ were calculated from the temperature dependence of the KIE measured below the observed breakpoint (−27 °C) (Fig. 3).

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FIGURE 3.

Temperature dependence of the rate constants for the hydride transfer step. Eyring plots of ln(k_obs/T) versus 1/T for the hydride transfer step with NADPH and pro-S-NADP²H (NADPD) are shown for POR from T. elongatus (A), Synechocystis sp. PCC6803 (B), G. violaceus (C), and A. thaliana POR B (D). Data are shown fitted to the Eyring equation. Activation enthalpies (ΔH^‡) and activation entropies (ΔS^‡) are shown in Table 1 and supplemental Table S1. The arrows indicate a conserved breakpoint at −27 °C, and error bars were calculated from the average of at least five kinetic traces. The insets show the dependence of ln KIE as a function of 1/T to illustrate how the KIE becomes essentially independent of temperature below the −27 °C breakpoint.

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FIGURE 5.

Evolution of dynamics in the hydride and proton transfer reactions catalyzed by POR. A, the degree of solvent-coupled dynamics required for the hydride and proton transfer steps for PORs that span the evolutionary spectrum. The relative levels of solvent-coupled dynamics were obtained by fitting the viscosity dependence of the hydride and proton transfer rates (supplemental Fig. S5) to Equation 1. The different POR enzymes are denoted by the following abbreviations: T. e., T. elongatus BP-1; Syn., Synechocystis sp. PCC6803; G. v., G. violaceus; and A. t., A. thaliana POR B. B, scheme illustrating the twin-track approach for the evolution of dynamics in POR. The light-driven hydride transfer step is controlled by localized dynamics in all PORs through conservation of protein structure across evolutionary history (“cliff-edge” effect). Conversely, the dynamics required for proton transfer have evolved gradually, and the chemistry exhibits less reliance on complex dynamic networks in plant PORs.

Diverse Dynamic Regimes Control Proton Transfer

In contrast to the hydride transfer reaction, significant differences exist in the dynamic control of the subsequent proton transfer reaction. Proton transfer is faster (in both H₂O and ²H₂O buffers) (supplemental Fig. S3), and there is a smaller accompanying solvent isotope effect (SIE) for Arabidopsis and Gloeobacter PORs compared with the other cyanobacterial enzymes (Table 1), consistent with a narrower reaction barrier that facilitates quantum mechanical tunneling. Eyring plots (Fig. 4) indicate that different models for dynamically coupled hydrogen tunneling (25, 26) are required to explain the significant differences observed in the temperature dependence of the proton transfer reactions. The SIE is temperature-dependent in the thermophilic and mesophilic PORs (Fig. 4 and supplemental Table S2), consistent with a proton tunneling reaction dependent on fast (subpicosecond) promoting motions coupled to the reaction coordinate (8, 12, 26, 28). The need for compression of the energy barrier for proton tunneling implies a less than optimal structural configuration for this reaction in the cyanobacterial POR enzymes. By contrast, the rate of proton transfer for the plant and Gloeobacter enzymes is less dependent on temperature, and there is no measurable temperature dependence on the SIE (Fig. 4 and supplemental Table S2). This suggests that the active site is optimally configured (or evolved) for proton transfer, as there is no detectable promoting motion and consequent reliance on barrier compression to support a high probability of tunneling (4, 28).

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FIGURE 4.

Temperature dependence of the rate constants for the proton transfer step. Eyring plots of ln(k_obs/T) versus 1/T for the proton transfer step in H₂O and D₂O are shown for POR from T. elongatus (A), Synechocystis sp. PCC6803 (B), G. violaceus (C), and A. thaliana POR B (D). Data are shown fitted to the Eyring equation. Activation enthalpies (ΔH^‡) and activation entropies (ΔS^‡) are shown in Table 1 and supplemental Table S2. Error bars were calculated from the average of at least five kinetic traces. The insets show the dependence of ln KIE as a function of 1/T.

Solvent fluctuations are important in controlling the dynamics of protein systems, and these can be explored through studies of the dependence of reaction rates on solvent viscosity (9, 21, 30,–32). We found major differences in the viscosity dependence of the proton transfer reaction for each of the POR enzymes, reflecting different extents of solvent control on the internal protein dynamics for this reaction (Fig. 5A and supplemental Fig. S4). The rate constant for proton transfer in the cyanobacterial enzymes is very sensitive to changes in the solvent viscosity, suggesting that a network of long-range solvent-coupled protein motions is required for proton transfer (9). In contrast, in the Gloeobacter enzyme, the solvent viscosity of the rate constant for proton transfer is significantly diminished, revealing a less dominant “slaving” effect of solvent dynamics on protein motions and reaction chemistry. Moreover, in the POR enzyme from Arabidopsis, the rate constant for proton transfer is not dependent on solvent, suggesting a small or no role for a network of long-range solvent-coupled protein motions. We infer that the faster proton transfers observed in the plant POR enzymes are reliant only on localized motions in the active site.

These differences point to considerable variation in the energy and conformational space explored by the active site proton donor (a tyrosine residue) (15) and the Pchlide substrate in different POR enzymes. Sequence analysis shows that the Pchlide-binding site is very well conserved in plant PORs but is more diverse in the cyanobacterial POR enzymes (Fig. 2). Hence, the less stringent geometrical and dynamic constraints on proton transfer compared with the highly endergonic hydride transfer have resulted in an optimal configuration for proton transfer in plant POR enzymes, whereas the cyanobacterial POR enzymes require significant protein rearrangements to ensure that an optimal proton donor-acceptor distance is achieved. This more dominant dynamic control of the proton transfer chemistry in cyanobacterial PORs also complements slower motions associated with product release and substrate binding in these enzymes. Laser experiments on the millisecond time scale indicate major conformational change associated with release of the chlorophyllide and NADP⁺ products from T. elongatus POR (18), but these are not observed in comparable studies with the Gloeobacter and Arabidopsis enzymes (supplemental Fig. S5 and supplemental Table S3).