Ultrafast enzymatic reaction dynamics in protochlorophyllide oxidoreductase

The reaction catalyzed by the light-driven enzyme protochlorophyllide oxidoreductase (POR) has been initiated with a 50-fs laser pulse. We show that the catalytic mechanism, involving proton and hydride transfers, proceeds with time constants of 3 ps and 400 ps. It is known that catalysis by POR involves thermally excited protein dynamics; our results show that these molecular motions occur on an ultrafast timescale.     

Mutagenesis Alters the Catalytic Mechanism of the Light-driven Enzyme Protochlorophyllide Oxidoreductase

The light-activated enzyme protochlorophyllide oxidoreductase (POR) catalyzes an essential step in the synthesis of the most abundant pigment on Earth, chlorophyll. This unique reaction involves the sequential addition of a hydride and proton across the C17=C18 double bond of protochlorophyllide (Pchlide) by dynamically coupled quantum tunneling and is an important model system for studying the mechanism of hydrogen transfer reactions. In the present work, we have combined site-directed mutagenesis studies with a variety of sensitive spectroscopic and kinetic measurements to provide new insights into the mechanistic role of three universally conserved Cys residues in POR. We show that mutation of Cys-226 dramatically alters the catalytic mechanism of the enzyme. In contrast to wild-type POR, the characteristic charge-transfer intermediate, formed upon hydride transfer from NADPH to the C17 position of Pchlide, is absent in C226S variant enzymes. This suggests a concerted hydrogen transfer mechanism where proton transfer only is rate-limiting. Moreover, Pchlide reduction does not require the network of solvent-coupled conformational changes that play a key role in the proton transfer step of wild-type POR. We conclude that this globally important enzyme is finely tuned to facilitate efficient photochemistry, and the removal of a key interaction with Pchlide in the C226S variants significantly affects the local active site structure in POR, resulting in a shorter donor-acceptor distance for proton transfer.     

Structural bases of hydrogen tunneling in enzymes: progress and puzzles

Accumulating experimental evidence suggests that the occurrence of hydrogen tunneling is likely to be widespread in enzyme-catalyzed reactions. The realization that hydrogen can transfer via tunneling mechanisms has far-reaching implications for our understanding of enzyme catalysis involving proton, hydride or hydrogen atom transfer reactions. The current status of the field is highlighted by three enzyme systems that have been under intensive study in recent years, including soybean lipoxygenase-1, thermophilic alcohol dehydrogenase and dihydrofolate reductase. Particular attention has been devoted to the issues of whether protein dynamics modulate hydrogen tunneling probability and whether the tunneling process contributes to the catalytic power of enzymes.     

Prolamellar bodies formed by cyanobacterial protochlorophyllide oxidoreductase in Arabidopsis

In angiosperms, chlorophyll biosynthesis is light dependent. A key factor in this process is protochlorophyllide oxidoreductase (POR), which requires light to catalyze the reduction of protochlorophyllide to chlorophyllide. It is believed that this protein originated from an ancient cyanobacterial enzyme that was introduced into proto‐plant cells during the primary symbiosis. Here we report that PORs from the cyanobacteria Gloeobacter violaceus PCC7421 and Synechocystis sp. PCC6803 function in plastids. First, we found that the G. violaceus POR shows a higher affinity to its substrate protochlorophyllide than the Synechocystis POR but a similar affinity to plant PORs. Secondly, the reduced size of prolamellar bodies caused by a knockdown mutation of one of the POR genes, PORA, in Arabidopsis could be complemented by heterologous expression of the cyanobacterial PORs. Photoactive protochlorophyllide in the etioplasts of the complementing lines, however, was retained at a low level as in the parent PORA knockdown mutant, indicating that the observed formation of prolamellar bodies was irrelevant to the assembly of photoactive protochlorophyllide. This work reveals a new view on the formation of prolamellar bodies and provides new clues about the function of POR in the etioplast–chloroplast transition.     

Amino acid residues in the nicotinamide binding site contribute to catalysis by horse liver alcohol dehydrogenase

Amino acid residues Thr-178, Val-203, and Val-292, which interact with the nicotinamide ring of the coenzyme bound to alcohol dehydrogenase (ADH), may facilitate hydride transfer and hydrogen tunneling by orientation and dynamic effects. The T178S, T178V, V203A, V292A, V292S, and V292T substitutions significantly alter the steady state and transient kinetics of the enzyme. The V292A, V292S, and V292T enzymes have decreased affinity for coenzyme (NAD+ by 30-50-fold and NADH by 35-75-fold) as compared to the wild-type enzyme. The substitutions in the nicotinamide binding site decrease the rate constant of hydride transfer for benzyl alcohol oxidation by 3-fold (for V292T ADH) to 16-fold (for V203A ADH). The modest effects suggest that catalysis does not depend critically on individual residues and that several residues in the nicotinamide binding site contribute to catalysis. The structures of the V292T ADH-NAD+-pyrazole and wild-type ADH-NAD+-4-iodopyrazole ternary complexes are very similar. Only subtle changes in the V292T enzyme cause the large changes in coenzyme binding and the small change in hydride transfer. In these complexes, one pyrazole nitrogen binds to the catalytic zinc, and the other nitrogen forms a partial covalent bond with C4 of the nicotinamide ring, which adopts a boat conformation that is postulated to be relevant for hydride transfer. The results provide an experimental basis for evaluating the contributions of dynamics to hydride transfer.     

Making light work of enzyme catalysis: protochlorophyllide oxidoreductase

In the chlorophyll biosynthetic pathway, the enzyme protochlorophyllide oxidoreductase (POR) catalyses a key light-driven reaction that triggers a profound transformation in plant development. Because POR is activated by light, it can provide information on the way in which light energy can be harnessed to power enzyme reactions and it presents us with a unique opportunity to study catalysis at low temperatures and on ultrafast timescales that are not accessible for most analyses of enzyme function. Recent advances in our understanding of the catalytic mechanism of POR illustrate why it is an important generic model for studying enzyme catalysis and reaction dynamics.     

Energy Landscapes and Catalysis in Nitric-oxide Synthase

Nitric oxide (NO) plays diverse roles in mammalian physiology. It is involved in blood pressure regulation, neurotransmission, and immune response, and is generated through complex electron transfer reactions catalyzed by NO synthases (NOS). In neuronal NOS (nNOS), protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the heme oxygenase domain, the site of NO generation. Simple models based on crystal structures of nNOS reductase have invoked a role for large scale motions of the FMN-binding domain in shuttling electrons from the FAD-binding domain to the heme oxygenase domain. However, molecular level insight of the dynamic structural transitions in NOS enzymes during enzyme catalysis is lacking. We use pulsed electron-electron double resonance spectroscopy to derive inter-domain distance relationships in multiple conformational states of nNOS. These distance relationships are correlated with enzymatic activity through variable pressure kinetic studies of electron transfer and turnover. The binding of NADPH and calmodulin are shown to influence interdomain distance relationships as well as reaction chemistry. An important effect of calmodulin binding is to suppress adventitious electron transfer from nNOS to molecular oxygen and thereby preventing accumulation of reactive oxygen species. A complex landscape of conformations is required for nNOS catalysis beyond the simple models derived from static crystal structures of nNOS reductase. Detailed understanding of this landscape advances our understanding of nNOS catalysis/electron transfer, and could provide new opportunities for the discovery of small molecule inhibitors that bind at dynamic protein interfaces of this multidimensional energy landscape.     

Mechanistic Reappraisal of Early Stage Photochemistry in the Light-Driven Enzyme Protochlorophyllide Oxidoreductase

The light-driven enzyme protochlorophyllide oxidoreductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide). This reaction is a key step in the biosynthesis of chlorophyll. Ultrafast photochemical processes within the Pchlide molecule are required for catalysis and previous studies have suggested that a short-lived excited-state species, known as I675*, is the first catalytic intermediate in the reaction and is essential for capturing excitation energy to drive subsequent hydride and proton transfers. The chemical nature of the I675* excited state species and its role in catalysis are not known. Here, we report time-resolved pump-probe spectroscopy measurements to study the involvement of the I675* intermediate in POR photochemistry. We show that I675* is not unique to the POR-catalyzed photoreduction of Pchlide as it is also formed in the absence of the POR enzyme. The I675* species is only produced in samples that contain both Pchlide substrate and Chlide product and its formation is dependent on the pump excitation wavelength. The rate of formation and the quantum yield is maximized in 50∶50 mixtures of the two pigments (Pchlide and Chlide) and is caused by direct energy transfer between Pchlide and neighboring Chlide molecules, which is inhibited in the polar solvent methanol. Consequently, we have re-evaluated the mechanism for early stage photochemistry in the light-driven reduction of Pchlide and propose that I675* represents an excited state species formed in Pchlide-Chlide dimers, possibly an excimer. Contrary to previous reports, we conclude that this excited state species has no direct mechanistic relevance to the POR-catalyzed reduction of Pchlide.     

Evolutionary Aspects of Enzyme Dynamics

The role of evolutionary pressure on the chemical step catalyzed by enzymes is somewhat enigmatic, in part because chemistry is not rate-limiting for many optimized systems. Herein, we present studies that examine various aspects of the evolutionary relationship between protein dynamics and the chemical step in two paradigmatic enzyme families, dihydrofolate reductases and alcohol dehydrogenases. Molecular details of both convergent and divergent evolution are beginning to emerge. The findings suggest that protein dynamics across an entire enzyme can play a role in adaptation to differing physiological conditions. The growing tool kit of kinetics, kinetic isotope effects, molecular biology, biophysics, and bioinformatics provides means to link evolutionary changes in structure-dynamics function to the vibrational and conformational states of each protein.     

Dynamic mechanism of proton transfer in mannitol 2-dehydrogenase from Pseudomonas fluorescens: mobile GLU292 controls proton relay through a water channel that connects the active site with bulk solvent

The active site of mannitol 2-dehydrogenase from Pseudomonas fluorescens (PfM2DH) is connected with bulk solvent through a narrow protein channel that shows structural resemblance to proton channels utilized by redox-driven proton pumps. A key element of the PfM2DH channel is the "mobile" Glu(292), which was seen crystallographically to adopt distinct positions up and down the channel. It was suggested that the "down → up" conformational change of Glu(292) could play a proton relay function in enzymatic catalysis, through direct proton shuttling by the Glu or because the channel is opened for water molecules forming a chain along which the protons flow. We report evidence from site-directed mutagenesis (Glu(292) → Ala) substantiated by data from molecular dynamics simulations that support a role for Glu(292) as a gate in a water chain (von Grotthuss-type) mechanism of proton translocation. Occupancy of the up and down position of Glu(292) is influenced by the bonding and charge state of the catalytic acid base Lys(295), suggesting that channel opening/closing motions of the Glu are synchronized to the reaction progress. Removal of gatekeeper control in the E292A mutant resulted in a selective, up to 120-fold slowing down of microscopic steps immediately preceding catalytic oxidation of mannitol, consistent with the notion that formation of the productive enzyme-NAD(+)-mannitol complex is promoted by a corresponding position change of Glu(292), which at physiological pH is associated with obligatory deprotonation of Lys(295) to solvent. These results underscore the important role of conformational dynamics in the proton transfer steps of alcohol dehydrogenase catalysis.     

Preservation of Protein Dynamics in Dihydrofolate Reductase Evolution

The hydride transfer reaction catalyzed by dihydrofolate reductase (DHFR) is a model for examining how protein dynamics contribute to enzymatic function. The relationship between functional motions and enzyme evolution has attracted significant attention. Recent studies on N23PP Escherichia coli DHFR (ecDHFR) mutant, designed to resemble parts of the human enzyme, indicated a reduced single turnover rate. NMR relaxation dispersion experiments with that enzyme showed rigidification of millisecond Met-20 loop motions (Bhabha, G., Lee, J., Ekiert, D. C., Gam, J., Wilson, I. A., Dyson, H. J., Benkovic, S. J., and Wright, P. E. (2011) Science 332, 234–238). A more recent study of this mutant, however, indicated that fast motions along the reaction coordinate are actually more dispersed than for wild-type ecDHFR (WT). Furthermore, a double mutant (N23PP/G51PEKN) that better mimics the human enzyme seems to restore both the single turnover rates and narrow distribution of fast dynamics (Liu, C. T., Hanoian, P., French, T. H., Hammes-Schiffer, S., and Benkovic, S. J. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 10159–11064). Here, we measured intrinsic kinetic isotope effects for both N23PP and N23PP/G51PEKN double mutant DHFRs over a temperature range. The findings indicate that although the C-H→C transfer and dynamics along the reaction coordinate are impaired in the altered N23PP mutant, both seem to be restored in the N23PP/G51PEKN double mutant. This indicates that the evolution of G51PEKN, although remote from the Met-20 loop, alleviated the loop rigidification that would have been caused by N23PP, enabling WT-like H-tunneling. The correlation between the calculated dynamics, the nature of C-H→C transfer, and a phylogenetic analysis of DHFR sequences are consistent with evolutionary preservation of the protein dynamics to enable H-tunneling from well reorganized active sites.     

Force-clamp spectroscopy detects residue co-evolution in enzyme catalysis

Understanding how the catalytic mechanisms of enzymes are optimized through evolution remains a major challenge in molecular biology. The concept of co-evolution implicates that compensatory mutations occur to preserve the structure and function of proteins. We have combined statistical analysis of protein sequences with the sensitivity of single molecule force-clamp spectroscopy to probe how catalysis is affected by structurally distant correlated mutations in Escherichia coli thioredoxin. Our findings show that evolutionary anti-correlated mutations have an inhibitory effect on enzyme catalysis, whereas positively correlated mutations rescue the catalytic activity. We interpret these results in terms of an evolutionary tuning of both the enzyme-substrate binding process and the chemistry of the active site. Our results constitute a direct observation of distant residue co-evolution in enzyme catalysis.     

Ultrafast catalytic processes in enzymes

The study of biocatalysis and biotransformation in the transition-state region has been challenging and difficult, but recent advances on two important photoenzymes in nature, DNA photolyase and protochlorophyllide oxidoreductase, have enabled the investigation of their catalytic processes in real time. By following the entire evolution of substrate transformation, the functional dynamics constituting a series of elementary reactions have been mapped out. The five fundamental reactions in the enzymes, namely electron transfer, bond breaking and making, proton and hydride transfer, all occur ultrafast within subnanosecond. The direct clocking of catalytic transition states probes central, unmasked chemical processes and provides mechanistic insights into the role of the dynamics in enzyme function, which not only facilitates the formation of the enzyme-substrate complex in the transition-state configurations, but also modulates the subsequent catalytic reactions for maximum biotransformation efficiency.     

A proton delivery pathway in the soluble fumarate reductase from Shewanella frigidimarina

The mechanism for fumarate reduction by the soluble fumarate reductase from Shewanella frigidimarina involves hydride transfer from FAD and proton transfer from the active-site acid, Arg-402. It has been proposed that Arg-402 forms part of a proton transfer pathway that also involves Glu-378 and Arg-381 but, unusually, does not involve any bound water molecules. To gain further insight into the importance of this proton pathway we have perturbed it by substituting Arg-381 by lysine and methionine and Glu-378 by aspartate. Although all the mutant enzymes retain measurable activities, there are orders-of-magnitude decreases in their k(cat) values compared with the wild-type enzyme. Solvent kinetic isotope effects show that proton transfer is rate-limiting in the wild-type and mutant enzymes. Proton inventories indicate that the proton pathway involves multiple exchangeable groups. Fast scan protein-film voltammetric studies on wild-type and R381K enzymes show that the proton transfer pathway delivers one proton per catalytic cycle and is not required for transporting the other proton, which transfers as a hydride from the reduced, protonated FAD. The crystal structures of E378D and R381M mutant enzymes have been determined to 1.7 and 2.1 A resolution, respectively. They allow an examination of the structural changes that disturb proton transport. Taken together, the results indicate that Arg-381, Glu-378, and Arg-402 form a proton pathway that is completely conserved throughout the fumarate reductase/succinate dehydrogenase family of enzymes.     

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.     

Molecular cloning and catalytic mechanism of a novel glycosphingolipid-degrading beta-N-acetylgalactosaminidase from Paenibacillus sp. TS12

We report here the molecular cloning, characterization, and catalytic mechanism of a novel glycosphingolipid-degrading β-N-acetylgalactosaminidase (β-NGA) from Paenibacillus sp. TS12 (NgaP). Consisting of 1034 putative amino acid residues, NgaP shares no sequence similarity with known proteins. Recombinant NgaP, expressed in Escherichia coli, cleaved the nonreducing terminal β-GalNAc residues of gangliotriaosylceramide and globotetraosylceramide. The enzyme hydrolyzed para-nitrophenyl-β-N-acetylgalactosaminide ～100 times faster than para-nitrophenyl-β-N-acetylglucosaminide. GalNAc thiazoline, an analog of the oxazolinium intermediate and potent inhibitor for enzymes adopting substrate-assisted catalysis, competitively inhibited the enzyme. The K(i) of the enzyme for GalNAc thiazoline was 1.3 nM, whereas that for GlcNAc thiazoline was 46.8 μM. Comparison of the secondary structure with those of known enzymes exhibiting substrate-assisted catalysis and point mutation analysis indicated that NgaP adopts substrate-assisted catalysis in which Glu-608 and Asp-607 could function as a proton donor and a stabilizer of the 2-acetamide group of the β-GalNAc at the active site, respectively. These results clearly indicate that NgaP is a β-NGA showing substrate-assisted catalysis. This is the first report describing the molecular cloning of a β-NGA adopting substrate-assisted catalysis.     

Isolation and analysis of the gene encoding the pyruvate-ferredoxin oxidoreductase of Desulfovibrio africanus, production of the recombinant enzyme in Escherichia coli, and effect of carboxy-terminal deletions on its stability.

Previous studies have shown that the pyruvate-ferredoxin oxidoreductase (POR) of the sulfate-reducing bacterium Desulfovibrio africanus is a homodimer that contains one thiamine pyrophosphate and three [4Fe-4S]2+/1+ centers/subunit. Interestingly, the enzyme isolated from a strictly anaerobic bacterium is highly stable in the presence of oxygen, in contrast to the other PORs characterized in anaerobic organisms (L. Pieulle, B. Guigliarelli, M. Asso, F. Dole, A. Bernadac, and E. C. Hatchikian, Biochim. Biophys. Acta 1250:49-59, 1995). We report here the determination of the nucleotide sequence of the por gene encoding the D. africanus POR. The amino acid sequence deduced from this nucleotide sequence corresponds to the first primary structure of a homodimeric POR from strictly anaerobic bacteria. The subunit of the D. africanus POR contains two ferredoxin-type [4Fe-4S] cluster binding motifs (CX2CX2CX3CP) and four additional highly conserved cysteines belonging to a nontypical motif. These 12 cysteine residues may coordinate the three Fe-S centers present in D. africanus POR. The thiamine pyrophosphate binding domain is located in the C-terminal part of the protein close to the four conserved cysteine residues. The D. africanus enzyme sequence appears homologous to the other POR sequences. However, the enzyme differs from all other PORs by a C-terminal extension of about 60 residues of its polypeptide chain. The two cysteine residues located in this additional region may be involved in the formation of a disulfide bridge associated with the activation process of the catalytic activity. The por gene has been expressed, for the first time, in anaerobically grown Escherichia coli behind the isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter, resulting in the production of POR in its active form. The recombinant enzyme is stable toward oxygen during several days, and initial characterization of the recombinant POR showed that its activity increased in the presence of dithioerythritol. These properties indicate that the recombinant POR behaves like the native D. africanus enzyme. The study of carboxy-terminal deletion mutants strongly suggests that deletions in the C-terminal region of D. africanus enzyme can have dramatic effects on the stability of the enzyme toward oxygen.     

Conformational changes and catalysis by alcohol dehydrogenase

As shown by X-ray crystallography, horse liver alcohol dehydrogenase undergoes a global conformational change upon binding of NAD⁺ or NADH, involving a rotation of the catalytic domain relative to the coenzyme binding domain and the closing up of the active site to produce a catalytically efficient enzyme. The conformational change requires a complete coenzyme and is affected by various chemical or mutational substitutions that can increase the catalytic turnover by altering the kinetics of the isomerization and rate of dissociation of coenzymes. The binding of NAD⁺ is kinetically limited by a unimolecular isomerization (corresponding to the conformational change) that is controlled by deprotonation of the catalytic zinc-water to produce a negatively-charged zinc-hydroxide, which can attract the positively-charged nicotinamide ring. The deprotonation is facilitated by His-51 acting through a hydrogen-bonded network to relay the proton to solvent. Binding of NADH also involves a conformational change, but the rate is very fast. After the enzyme binds NAD⁺ and closes up, the substrate displaces the hydroxide bound to the catalytic zinc; this exchange may involve a double displacement reaction where the carboxylate group of a glutamate residue first displaces the hydroxide (inverting the tetrahedral coordination of the zinc), and then the exogenous ligand displaces the glutamate. The resulting enzyme-NAD⁺-alcoholate complex is poised for hydrogen transfer, and small conformational fluctuations may bring the reactants together so that the hydride ion is transferred by quantum mechanical tunneling. In the process, the nicotinamide ring may become puckered, as seen in structures of complexes of the enzyme with NADH. The conformational changes of alcohol dehydrogenase demonstrate the importance of protein dynamics in catalysis.     

The first catalytic step of the light-driven enzyme protochlorophyllide oxidoreductase proceeds via a charge transfer complex

In chlorophyll biosynthesis protochlorophyllide reductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide) to chlorophyllide, providing a rare opportunity to trap and characterize catalytic intermediates at low temperatures. Moreover, the presence of a chlorophyll-like molecule allows the use of EPR, electron nuclear double resonance, and Stark spectroscopies, previously used for the analysis of photosynthetic systems, to follow catalytic events in the active site of POR. Different models involving the formation of either radical species or charge transfer complexes have been proposed for the initial photochemical step, which forms a nonfluorescent intermediate absorbing at 696 nm (A696). Our EPR data show that the concentration of the radical species formed in the initial photochemical step is not stoichiometric with conversion of substrate. Instead, a large Stark effect, indicative of charge transfer character, is associated with A696. Two components were required to fit the Stark data, providing clear evidence that charge transfer complexes are formed during the initial photochemistry. The temperature dependences of both A696 formation and NADPH oxidation are identical, and we propose that formation of the A696 state involves hydride transfer from NADPH to form a charge transfer complex. A catalytic mechanism of POR is suggested in which Pchlide absorbs a photon, creating a transient charge separation across the C-17-C-18 double bond, which promotes ultrafast hydride transfer from the pro-S face of NADPH to the C-17 of Pchlide. The resulting A696 charge transfer intermediate facilitates transfer of a proton to the C-18 of Pchlide during the subsequent first "dark" reaction.     

The role of protein surface charge in catalytic activity and chloroplast membrane association of the pea NADPH: protochlorophyllide oxidoreductase (POR) as revealed by alanine scanning mutagenesis

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide (pchlide) to chlorophyllide (chlide) in the biosynthesis of chlorophyll. POR is a peripheral membrane protein that accumulates to high levels in the prolamellar bodies of vascular plant etioplasts and is present at low levels in the thylakoid membranes of developing and mature plastids. Clustered charged-to-alanine scanning mutagenesis of the pea (Pisum sativum L.) POR was carried out and the resulting mutant enzymes analyzed for their ability to catalyze pchlide photoconversion in vivo and to associate properly with thylakoid membrane preparations in vitro. Of 37 mutant enzymes examined, 5 retained wild-type levels of activity, 14 were catalytically inactive, and the remaining 18 exhibited altered levels of function. Several of the mutant enzymes showed temperature- dependent enzymatic activity, being inactive at 32°C, but partially active at 24°C. Mutations in predicted - helical regions of the protein showed the least effect on enzyme activity, whereas mutations in predicted -sheet regions of the protein showed a consistent adverse affect on enzyme function. In the absence of added NADPH, neither wild-type POR nor any of the mutant PORs resisted proteolysis by thermolysin following assembly onto the thylakoid membranes. In contrast, when NADPH was present in the assay mixture, 13 of the 37 mutant PORs examined were found to be resistant to thermolysin upon treatment, suggesting that the mutations did not affect their ability to be properly attached to the thylakoid membrane. In general, the replacement of charged amino acids by alanine in the most N- and C-terminal regions of the mature protein did not significantly affect POR assembly, whereas mutations within the central core of the protein (between residues 86 and 342) were incapable of proper attachment to the thylakoid. Failure to properly associate with the thylakoid membrane in a protease resistant manner was only weakly correlated to loss of catalytic function. These studies are a first step towards defining structural determinants crucial to POR function and intraorganellar localization.