Redox conditions correlated with vibronic coupling modulate quantum beats in photosynthetic pigment-protein complexes.

Quantum coherences, observed as time-dependent beats in ultrafast spectroscopic experiments, arise when light-matter interactions prepare systems in superpositions of states with differing energy and fixed phase across the ensemble. Such coherences have been observed in photosynthetic systems following ultrafast laser excitation, but what these coherences imply about the underlying energy transfer dynamics remains subject to debate. Recent work showed that redox conditions tune vibronic coupling in the Fenna-Matthews-Olson (FMO) pigment-protein complex in green sulfur bacteria, raising the question of whether redox conditions may also affect the long-lived (>100 fs) quantum coherences observed in this complex. In this work, we perform ultrafast two-dimensional electronic spectroscopy measurements on the FMO complex under both oxidizing and reducing conditions. We observe that many excited-state coherences are exclusively present in reducing conditions and are absent or attenuated in oxidizing conditions. Reducing conditions mimic the natural conditions of the complex more closely. Further, the presence of these coherences correlates with the vibronic coupling that produces faster, more efficient energy transfer through the complex under reducing conditions. The growth of coherences across the waiting time and the number of beating frequencies across hundreds of wavenumbers in the power spectra suggest that the beats are excited-state coherences with a mostly vibrational character whose phase relationship is maintained through the energy transfer process. Our results suggest that excitonic energy transfer proceeds through a coherent mechanism in this complex and that the coherences may provide a tool to disentangle coherent relaxation from energy transfer driven by stochastic environmental fluctuations.

Photosynthesis tunes quantum-mechanical mixing of electronic and vibrational states to steer exciton energy transfer.

Photosynthetic species evolved to protect their light-harvesting apparatus from photoxidative damage driven by intracellular redox conditions or environmental conditions. The Fenna-Matthews-Olson (FMO) pigment-protein complex from green sulfur bacteria exhibits redox-dependent quenching behavior partially due to two internal cysteine residues. Here, we show evidence that a photosynthetic complex exploits the quantum mechanics of vibronic mixing to activate an oxidative photoprotective mechanism. We use two-dimensional electronic spectroscopy (2DES) to capture energy transfer dynamics in wild-type and cysteine-deficient FMO mutant proteins under both reducing and oxidizing conditions. Under reducing conditions, we find equal energy transfer through the exciton 4-1 and 4-2-1 pathways because the exciton 4-1 energy gap is vibronically coupled with a bacteriochlorophyll-a vibrational mode. Under oxidizing conditions, however, the resonance of the exciton 4-1 energy gap is detuned from the vibrational mode, causing excitons to preferentially steer through the indirect 4-2-1 pathway to increase the likelihood of exciton quenching. We use a Redfield model to show that the complex achieves this effect by tuning the site III energy via the redox state of its internal cysteine residues. This result shows how pigment-protein complexes exploit the quantum mechanics of vibronic coupling to steer energy transfer.

The influence of quaternary structure on the stability of Fenna-Matthews-Olson (FMO) antenna complexes.

The trimeric nature of the Fenna-Matthews-Olson (FMO) protein antenna complex from green sulfur phototrophic bacteria was investigated. Mutations were introduced into the protein at positions 142 and 198, which were chosen to destabilize the intra-trimer salt bridges between adjacent monomers. Strains bearing the mutations R142L, R198L, or their combination, exhibited altered optical absorption spectra of purified membranes and fluoresced more intensely than the wild type. In particular, the introduction of the R142L mutation resulted in slower culture growth rates, as well as an FMO complex that was not able to be isolated in appreciable quantities, while the R198L mutation yielded an FMO complex with increased sensitivity to sodium thiocyanate and Triton X-100 treatments. Native and denaturing PAGE experiments suggest that much of the FMO complexes in the mutant strains pool with the insoluble material upon membrane solubilization with n-dodecyl ß-D-maltoside, a mild nonionic detergent. Taken together, our results suggest that the quaternary structure of the FMO complex, the homotrimer, is an important factor in the maintenance of the complex's tertiary structure.

Evidence for a cysteine-mediated mechanism of excitation energy regulation in a photosynthetic antenna complex.

Light-harvesting antenna complexes not only aid in the capture of solar energy for photosynthesis, but regulate the quantity of transferred energy as well. Light-harvesting regulation is important for protecting reaction center complexes from overexcitation, generation of reactive oxygen species, and metabolic overload. Usually, this regulation is controlled by the association of light-harvesting antennas with accessory quenchers such as carotenoids. One antenna complex, the Fenna-Matthews-Olson (FMO) antenna protein from green sulfur bacteria, completely lacks carotenoids and other known accessory quenchers. Nonetheless, the FMO protein is able to quench energy transfer in aerobic conditions effectively, indicating a previously unidentified type of regulatory mechanism. Through de novo sequencing MS, chemical modification, and mutagenesis, we have pinpointed the source of the quenching action to cysteine residues (Cys49 and Cys353) situated near two low-energy bacteriochlorophylls in the FMO protein from Chlorobaculum tepidum Removal of these cysteines (particularly removal of the completely conserved Cys353) through N-ethylmaleimide modification or mutagenesis to alanine abolishes the aerobic quenching effect. Electrochemical analysis and electron paramagnetic resonance spectra suggest that in aerobic conditions the cysteine thiols are converted to thiyl radicals which then are capable of quenching bacteriochlorophyll excited states through electron transfer photochemistry. This simple mechanism has implications for the design of bio-inspired light-harvesting antennas and the redesign of natural photosynthetic systems.

Energy landscape of the intact and destabilized FMO antennas from C. tepidum and the L122Q mutant: Low temperature spectroscopy and modeling study.

We discuss the excitonic energy landscape of the typically studied wild-type (WT) Fenna-Matthews-Olson (FMO) antenna protein from the green sulfur bacterium Chlorobaculum tepidum (referred to as WTM), which is described as a mixture of intact (WTI) and destabilized (WTD) complexes. Optical spectra of WTM and the L122Q mutant (where leucine 122 near BChl 8 is replaced with glutamine) are compared to WTI FMO. We show that WTM and L122Q samples are mixtures of two subpopulations of proteins, most likely induced by protein conformational changes during the isolation/purification procedures. Absorption, emission, and HB spectra of WTM and L122Q mutant are very similar, in which the low-energy trap (revealed by the nonresonant HB spectra) shifts to higher energies as a function of fluence, supporting a mixture model. No fluence-dependent shift is observed in the WTI FMO trimers. New Hamiltonians are provided for WTI and WTD proteins. Resonant HB spectra show that the internal energy relaxation times in the WTM and L122Q mutant are similar, and depend on excitation frequency. Fast average relaxation times (excited state lifetimes) are observed for burning into the main broad absorption band near 805nm. Burning at longer wavelengths reveals slower total dephasing times. No resonant bleach is observed at λB≤803nm, implying much faster (femtosecond) energy relaxation in this spectral range in agreement with 2D electronic spectroscopy frequency maps.

Photoactivation and relaxation studies on the cyanobacterial orange carotenoid protein in the presence of copper ion.

Photosynthesis starts with absorption of light energy by light-harvesting antenna complexes with subsequent production of energy-rich organic compounds. However, all photosynthetic organisms face the challenge of excess photochemical conversion capacity. In cyanobacteria, non-photochemical quenching (NPQ) performed by the orange carotenoid protein (OCP) is one of the most important mechanisms to regulate the light energy captured by light-harvesting antennas. This regulation permits the cell to meet its cellular energy requirements and at the same time protects the photosynthetic apparatus under fluctuating light conditions. Several reports have revealed that thermal dissipation increases under excess copper in plants. To explore the effects and mechanisms of copper on cyanobacteria NPQ, photoactivation and relaxation of OCP in the presence of copper were examined in this communication. When OCPo (OCP at orange state) is converted into OCPr(OCP at red state), copper ion has no effect on the photoactivation kinetics. Relaxation of OCPr to OCPo, however, is largely delayed-almost completely blocked, in the presence of copper. Even the addition of the fluorescence recovery protein (FRP) cannot activate the relaxation process. Native polyacrylamide gel electrophoresis (PAGE) analysis result indicates the heterogeneous population of Cu2+-locked OCPr. The Cu2+-OCP binding constant was estimated using a hyperbolic binding curve. Functional roles of copper-binding OCP in vivo are discussed.

Light harvesting in phototrophic bacteria: structure and function.

This review serves as an introduction to the variety of light-harvesting (LH) structures present in phototrophic prokaryotes. It provides an overview of the LH complexes of purple bacteria, green sulfur bacteria (GSB), acidobacteria, filamentous anoxygenic phototrophs (FAP), and cyanobacteria. Bacteria have adapted their LH systems for efficient operation under a multitude of different habitats and light qualities, performing both oxygenic (oxygen-evolving) and anoxygenic (non-oxygen-evolving) photosynthesis. For each LH system, emphasis is placed on the overall architecture of the pigment-protein complex, as well as any relevant information on energy transfer rates and pathways. This review addresses also some of the more recent findings in the field, such as the structure of the CsmA chlorosome baseplate and the whole-cell kinetics of energy transfer in GSB, while also pointing out some areas in need of further investigation.

A Molecular Mechanism for Nonphotochemical Quenching in Cyanobacteria.

The cyanobacterial orange carotenoid protein (OCP) protects photosynthetic cyanobacteria from photodamage by dissipating excess excitation energy collected by phycobilisomes (PBS) as heat. Dissociation of the PBS-OCP complex in vivo is facilitated by another protein known as the fluorescence recovery protein (FRP), which primarily exists as a dimeric complex. We used various mass spectrometry (MS)-based techniques to investigate the molecular mechanism of this FRP-mediated process. FRP in the dimeric state (dFRP) retains its high affinity for the C-terminal domain (CTD) of OCP in the red state (OCPr). Site-directed mutagenesis and native MS suggest the head region on FRP is a candidate to bind OCP. After attachment to the CTD, the conformational changes of dFRP allow it to bridge the two domains, facilitating the reversion of OCPr into the orange state (OCPo) accompanied by a structural rearrangement of dFRP. Interestingly, we found a mutual response between FRP and OCP; that is, FRP and OCPr destabilize each other, whereas FRP and OCPo stabilize each other. A detailed mechanism of FRP function is proposed on the basis of the experimental results.

Native Mass Spectrometry Analysis of Oligomerization States of Fluorescence Recovery Protein and Orange Carotenoid Protein: Two Proteins Involved in the Cyanobacterial Photoprotection Cycle.

The orange carotenoid protein (OCP) and fluorescence recovery protein (FRP) are present in many cyanobacteria and regulate an essential photoprotection cycle in an antagonistic manner as a function of light intensity. We characterized the oligomerization states of OCP and FRP by using native mass spectrometry, a technique that has the capability of studying native proteins under a wide range of protein concentrations and molecular masses. We found that dimeric FRP is the predominant state at protein concentrations ranging from 3 to 180 µM and that higher-order oligomers gradually form at protein concentrations above this range. The OCP, however, demonstrates significantly different oligomerization behavior. Monomeric OCP (mOCP) dominates at low protein concentrations, with an observable population of dimeric OCP (dOCP). The ratio of dOCP to mOCP, however, increases proportionally with protein concentration. Higher-order OCP oligomers form at protein concentrations beyond 10 µM. Additionally, native mass spectrometry coupled with ion mobility allowed us to measure protein collisional cross sections and interrogate the unfolding of different FRP and OCP oligomers. We found that monomeric FRP exhibits a one-stage unfolding process, which could be correlated with its C-terminal bent crystal structure. The structural domain compositions of FRP and OCP are compared and discussed.

Probing the excitonic landscape of the Chlorobaculum tepidum Fenna-Matthews-Olson (FMO) complex: a mutagenesis approach.

In this paper we report the steady-state optical properties of a series of site-directed mutants in the Fenna-Matthews-Olson (FMO) complex of Chlorobaculum tepidum, a photosynthetic green sulfur bacterium. The FMO antenna complex has historically been used as a model system for energy transfer due to the water-soluble nature of the protein, its stability at room temperature, as well as the availability of high-resolution structural data. Eight FMO mutants were constructed with changes in the environment of each of the bacteriochlorophyll a pigments found within each monomer of the homotrimeric FMO complex. Our results reveal multiple changes in low temperature absorption, as well as room temperature CD in each mutant compared to the wild-type FMO complex. These datasets were subsequently used to model the site energies of each pigment in the FMO complex by employing three different Hamiltonians from the literature. This enabled a basic approximation of the site energy shifts imparted on each pigment by the changed amino acid residue. These simulations suggest that, while the three Hamiltonians used in this work provide good fits to the wild-type FMO absorption spectrum, further efforts are required to obtain good fits to the mutant minus wild-type absorption difference spectra. This demonstrates that the use of FMO mutants can be a valuable tool to refine and iterate the current models of energy transfer in this system.

Perturbation of bacteriochlorophyll molecules in Fenna-Matthews-Olson protein complexes through mutagenesis of cysteine residues.

The Fenna-Matthews-Olson (FMO) pigment-protein complex in green sulfur bacteria transfers excitation energy from the chlorosome antenna complex to the reaction center. In understanding energy transfer in the FMO protein, the individual contributions of the bacteriochlorophyll pigments to the FMO complex's absorption spectrum could provide detailed information with which molecular and energetic models can be constructed. The absorption properties of the pigments, however, are such that their spectra overlap significantly. To overcome this, we used site-directed mutagenesis to construct a series of mutant FMO complexes in the model green sulfur bacterium Chlorobaculum tepidum (formerly Chlorobium tepidum). Two cysteines at positions 49 and 353 in the C. tepidum FMO complex, which reside near hydrogen bonds between BChls 2 and 3, and their amino acid binding partner serine 73 and tyrosine 15, respectively, were changed to alanine residues. The resulting C49A, C353A, and C49A C353A double mutants were analyzed with a combination of optical absorption and circular dichroism (CD) spectroscopies. Our results revealed changes in the absorption properties of several underlying spectral components in the FMO complex, as well as the redox behavior of the complex in response to the reductant sodium dithionite. A high-resolution X-ray structure of the C49A C353A double mutant reveals that these spectral changes appear to be independent of any major structural rearrangements in the FMO mutants. Our findings provide important tests for theoretical calculations of the C. tepidum FMO absorption spectrum, and additionally highlight a possible role for cysteine residues in the redox activity of the pigment-protein complex.

Electron Transfer in Bacterial Reaction Centers with the Photoactive Bacteriopheophytin Replaced by a Bacteriochlorophyll through Coordinating Ligand Substitution.

The influence of amino acid substitutions at position M214 (M-subunit, residue 214) on the rate and pathway of electron transfer involving the bacteriopheophytin cofactor, HA, in a bacterial photosynthetic reaction center has been explored in a series of Rhodobacter sphaeroides mutants. The M214 leucine (L) residue of the wild type was replaced with histidine (H), glutamine (Q), and asparagine (N), creating the mutants M214LH, M214LQ, and M214LN, respectively. As has been reported previously for M214LH, each of these mutations resulted in a bacteriochlorophyll molecule in place of a bacteriopheophytin in the HA pocket, forming so-called ß-type mutants (in which the HA cofactor is called ßA). In addition, these mutations changed the properties of the surrounding protein environment in terms of charge distribution and the amino acid side chain volume. Electron transfer reactions from the excited primary donor P to the acceptor QA were characterized using ultrafast transient absorption spectroscopic techniques. Similar to that of the previously characterized M214LH (ß mutant), the strong energetic mixing of the P(+)BA(-) and P(+)ßA(-) states (the mixed anion is denoted I(-)) increased the rate of charge recombination between P(+) and I(-) in competition with the I(-) → QA forward reaction. This reduced the overall yield of charge separation forming the P(+)QA(-) state. While the kinetics of the primary electron transfer forming P(+)I(-) were essentially identical in all three ß mutants, the rates of the ßA(-) (I(-)) → QA electron transfer in M214LQ and M214LH were very similar but quite different from that of the M214LN mutant. The observed yield changes and the differences in kinetics are correlated more closely with the volume of the mutated amino acid than with their charge characteristics. These results are consistent with those of previous studies of a series of M214 mutants with different sizes of amino acid side chains that did not alter the HA cofactor composition [Pan, J., et al. (2013) J. Phys. Chem. B 117, 7179-7189]. Both studies indicate that protein relaxation in this region of the reaction center plays a key role in stabilizing charge-separated states involving the HA or ßA cofactor. The effect is particularly pronounced for reactions occurring on time scales of tens and hundreds of picoseconds (forward transfer to the QA and charge recombination).

Top-Down Mass Spectrometry Analysis of Membrane-Bound Light-Harvesting Complex 2 from Rhodobacter sphaeroides.

We report a top-down proteomic analysis of the membrane-bound peripheral light-harvesting complex LH2 isolated from the purple photosynthetic bacterium Rhodobacter sphaeroides. The LH2 complex is coded for by the puc operon. The Rb. sphaeroides genome contains two puc operons, designated puc1BAC and puc2BA. Although previous work has shown consistently that the LH2 ß polypeptide coded by the puc2B gene was assembled into LH2 complexes, there are contradictory reports as to whether the Puc2A polypeptides are incorporated into LH2 complexes. Furthermore, post-translational modifications of this protein offer the prospect that it could coordinate bacteriochlorophyll a (Bchl a) by a modified N-terminal residue. Here, we describe the components of the LH2 complex on the basis of electron-capture dissociation fragmentation to confirm the identity and sequence of the protein's subunits. We found that both gene products of the ß polypeptides are expressed and assembled in the mature LH2 complex, but only the Puc1A-encoded polypeptide α is observed here. The methionine of the Puc2B-encoded polypeptide is missing, and a carboxyl group is attached to the threonine at the N-terminus. Surprisingly, one amino acid encoded as an isoleucine in both the puc2B gene and the mRNA is found as valine in the mature LH2 complex, suggesting an unexpected and unusual post-translational modification or a specific tRNA recoding of this one amino acid.

Structural and kinetic properties of Rhodobacter sphaeroides photosynthetic reaction centers containing exclusively Zn-coordinated bacteriochlorophyll as bacteriochlorin cofactors.

The Zn-BChl-containing reaction center (RC) produced in a bchD (magnesium chelatase) mutant of Rhodobacter sphaeroides assembles with six Zn-bacteriochlorophylls (Zn-BChls) in place of four Mg-containing bacteriochlorophylls (BChls) and two bacteriopheophytins (BPhes). This protein presents unique opportunities for studying biological electron transfer, as Zn-containing chlorins can exist in 4-, 5-, and (theoretically) 6-coordinate states within the RC. In this paper, the electron transfer perturbations attributed exclusively to coordination state effects are separated from those attributed to the presence, absence, or type of metal in the bacteriochlorin at the HA pocket of the RC. The presence of a 4-coordinate Zn(2+) ion in the HA bacteriochlorin instead of BPhe results in a small decrease in the rates of the P*→P(+)HA(-)→P(+)QA(-) electron transfer, and the charge separation yield is not greatly perturbed; however coordination of the Zn(2+) by a fifth ligand provided by a histidine residue results in a larger rate decrease and yield loss. We also report the first crystal structure of a Zn-BChl-containing RC, confirming that the HA Zn-BChl was either 4- or 5-coordinate in the two types of Zn-BChl-containing RCs studied here. Interestingly, a large degree of disorder, in combination with a relatively weak anomalous difference electron density was found in the HB pocket. These data, in combination with spectroscopic results, indicate partial occupancy of this binding pocket. These findings provide insights into the use of BPhe as the bacteriochlorin pigment of choice at HA in both BChl- and Zn-BChl-containing RCs found in nature.

A DNA-directed light-harvesting/reaction center system.

A structurally and compositionally well-defined and spectrally tunable artificial light-harvesting system has been constructed in which multiple organic dyes attached to a three-arm-DNA nanostructure serve as an antenna conjugated to a photosynthetic reaction center isolated from Rhodobacter sphaeroides 2.4.1. The light energy absorbed by the dye molecules is transferred to the reaction center, where charge separation takes place. The average number of DNA three-arm junctions per reaction center was tuned from 0.75 to 2.35. This DNA-templated multichromophore system serves as a modular light-harvesting antenna that is capable of being optimized for its spectral properties, energy transfer efficiency, and photostability, allowing one to adjust both the size and spectrum of the resulting structures. This may serve as a useful test bed for developing nanostructured photonic systems.

Reengineering the optical absorption cross-section of photosynthetic reaction centers.

Engineered cysteine residues near the primary electron donor (P) of the reaction center from the purple photosynthetic bacterium Rhodobacter sphaeroides were covalently conjugated to each of several dye molecules in order to explore the geometric design and spectral requirements for energy transfer between an artificial antenna system and the reaction center. An average of 2.5 fluorescent dye molecules were attached at specific locations near P. The enhanced absorbance cross-section afforded by conjugation of Alexa Fluor 660 dyes resulted in a 2.2-fold increase in the formation of reaction center charge-separated state upon intensity-limited excitation at 650 nm. The effective increase in absorbance cross-section resulting from the conjugation of two other dyes, Alexa Fluor 647 and Alexa Fluor 750, was also investigated. The key parameters that dictate the efficiency of dye-to-reaction center energy transfer and subsequent charge separation were examined using both steady-state and time-resolved fluorescence spectroscopy as well as transient absorbance spectroscopy techniques. An understanding of these parameters is an important first step toward developing more complex model light-harvesting systems integrated with reaction centers.

Nano-mechanical mapping of the interactions between surface-bound RC-LH1-PufX core complexes and cytochrome c 2 attached to an AFM probe.

Electron transfer pathways in photosynthesis involve interactions between membrane-bound complexes such as reaction centres with an extrinsic partner. In this study, the biological specificity of electron transfer between the reaction centre-light-harvesting 1-PufX complex and its extrinsic electron donor, cytochrome c 2, formed the basis for mapping the location of surface-attached RC-LH1-PufX complexes using atomic force microscopy (AFM). This nano-mechanical mapping method used an AFM probe functionalised with cyt c 2 molecules to quantify the interaction forces involved, at the single-molecule level under native conditions. With surface-bound RC-His12-LH1-PufX complexes in the photo-oxidised state, the mean interaction force with cyt c 2 is approximately 480 pN with an interaction frequency of around 66 %. The latter value lowered 5.5-fold when chemically reduced RC-His12-LH1-PufX complexes are imaged in the dark to abolish electron transfer from cyt c 2 to the RC. The correspondence between topographic and adhesion images recorded over the same area of the sample shows that affinity-based AFM methods are a useful tool when topology alone is insufficient for spatially locating proteins at the surface of photosynthetic membranes.

Role of Rhodobacter sphaeroides photosynthetic reaction center residue M214 in the composition, absorbance properties, and conformations of H(A) and B(A) cofactors.

In the native reaction center (RC) of Rhodobacter sphaeroides, the side chain of (M)L214 projects orthogonally toward the plane and into the center of the A branch bacteriopheophytin (BPhe) macrocycle. The possibility that this side chain is responsible for the dechelation of the central Mg(2+) of bacteriochlorophyll (BChl) was investigated by replacement of (M)214 with residues possessing small, nonpolar side chains that can neither coordinate nor block access to the central metal ion. The (M)L214 side chain was also replaced with Cys, Gln, and Asn to evaluate further the requirements for assembly of the RC with BChl in the HA pocket. Photoheterotrophic growth studies showed no difference in growth rates of the (M)214 nonpolar mutants at a low light intensity, but the growth of the amide-containing mutants was impaired. The absorbance spectra of purified RCs indicated that although absorbance changes are associated with the nonpolar mutations, the nonpolar mutant RC pigment compositions are the same as in the wild-type protein. Crystal structures of the (M)L214G, (M)L214A, and (M)L214N mutants were determined (determined to 2.2-2.85 Å resolution), confirming the presence of BPhe in the HA pocket and revealing alternative conformations of the phytyl tail of the accessory BChl in the BA site of these nonpolar mutants. Our results demonstrate that (i) BChl is converted to BPhe in a manner independent of the aliphatic side chain length of nonpolar residues replacing (M)214, (ii) BChl replaces BPhe if residue (M)214 has an amide-bearing side chain, (iii) (M)214 side chains containing sulfur are not sufficient to bind BChl in the HA pocket, and (iv) the (M)214 side chain influences the conformation of the phytyl tail of the BA BChl.

Porphyrin Excretion Resulting From Mutation of a Gene Encoding a Class I Fructose 1,6-Bisphosphate Aldolase in Rhodobacter capsulatus.

This paper describes a mutant (called SB1707) of the Rhodobacter capsulatus wild type strain SB1003 in which a transposon-disrupted rcc01707 gene resulted in a ∼25-fold increase in the accumulation of coproporphyrin III in the medium of phototrophic (anaerobic) cultures grown in a yeast extract/peptone medium. There was little or no stimulation of pigment accumulation in aerobic cultures. Therefore, this effect of rcc01707 mutation appears to be specific for the anaerobic coproporphyrinogen III oxidase HemN as opposed to the aerobic enzyme HemF. The protein encoded by rcc01707 is homologous to Class I fructose 1,6-bisphosphate aldolases, which catalyze a glycolytic reaction that converts fructose 1, 6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, precursors of pyruvate. There were significant differences in coproporphyrin III accumulation using defined media with individual organic acids and sugars as the sole carbon source: pyruvate, succinate and glutamate stimulated accumulation the most, whereas glucose suppressed coproporphyrin III accumulation to 10% of that of succinate. However, although quantitatively lesser, similar effects of carbon source on the amount of accumulated pigment in the culture medium were seen in a wild type control. Therefore, this mutation appears to exaggerate effects also seen in the wild type strain. It is possible that mutation of rcc01707 causes a metabolic bottleneck or imbalance that was not rectified during growth on the several carbon sources tested. However, we speculate that, analogous to other fructose 1,6-bisphosphate aldolases, the rcc01707 gene product has a "moonlighting" activity that in this case is needed for the maximal expression of the hemN gene. Indeed, it was found that the rcc01707 gene is needed for maximal expression of a hemN promoter-lacZ reporter. With the decrease in hemN expression due to the absence of the rcc01707 gene product, coproporphyrinogen III accumulates and is released from the cell, yielding the spontaneous oxidation product coproporphyrin III.