Algae have adopted two primary strategies to maximize the performance of Rubisco in photosynthetic CO2 fixation. This has included either the development of a CO2-concentrating mechanism (CCM), based at the level of the chloroplast, or the evolution of the kinetic properties of Rubisco. This review examines the potential diversity of both Rubisco and chloroplast-based CCMs across algal divisions, including both green and nongreen algae, and seeks to highlight recent advances in our understanding of the area and future areas for research. Overall, the available data show that Rubisco enzymes from algae have evolved a higher affinity for CO2 when the algae have adopted a strategy for CO2 fixation that does not utilise a CCM. This appears to be true of both Green and Red Form I Rubisco enzymes found in green and nongreen algae, respectively. However, the Red Form I Rubisco enzymes present in nongreen algae appear to have reduced oxygenase potential at air level of O2. This has resulted in a photosynthetic physiology with a reduced potential to be inhibited by O2 and a reduced need to deal with photorespiration. In the limited number of microalgae that have been examined, there is a strong correlation between the existence of a high-affinity CCM physiology and the presence of pyrenoids in all algae, highlighting the potential importance of these chloroplast Rubisco-containing bodies. However, in macroalgae, there is greater diversity in the apparent relationships between pyrenoids and chloroplast features and the CCM physiology that the species shows. There are many examples of microalgae and macroalgae with variations in the presence and absence of pyrenoids as well as single and multiple chloroplasts per cell. This occurs in both green and nongreen algae and should provide ample material for extending studies in this area. Future research into the function of the pyrenoid and other chloroplast features, such as thylakoids, in the operation of a chloroplast-based CCM needs to be addressed in a diverse range of algal species. This should be approached together with assessment of the coevolution of Rubisco, particularly the evolution of Red Form I Rubisco enzymes, which appear to achieve superior kinetic characteristics when compared with the Rubisco of C3 higher plants, which are derived from green algal ancestors.Key words: Rubisco, CO2-concentrating mechanism, carbonic anhydrase, aquatic photosynthesis, algae, pyrenoids, inorganic carbon.

The crystal structure of dimeric Fe(III) superoxide dismutase (SOD) from Escherichia coli (3006 protein atoms, 2 irons, and 281 solvents) has been refined to an R of 0.184 using all observed data between 40.0 and 1.85 A (34,879 reflections). Features of this structure are compared with the refined structure of MnSOD from Thermus thermophilus. The coordination geometry at the Fe site is distorted trigonal bipyramidal, with axial ligands His26 and solvent (proposed to be OH-), and in-plane ligands His73, Asp156, and His160. Reduction of crystals to the Fe(II) state does not result in significant changes in metal-ligand geometry (R = 0.188 for data between 40.0 and 1.80 A). The arrangement of iron ligands in Fe(II) and Fe(III)SOD closely matches the Mn coordination found in MnSOD from T. thermophilus [Ludwig, M.L., Metzger, A.L., Pattridge, K.A., & Stallings, W.C. (1991) J. Mol. Biol. 219, 335-358]. Structures of the Fe(III) azide (40.0-1.8 A, R = 0.186) and Mn(III) azide (20.0-1.8 A, R = 0.179) complexes, reported here, reveal azide bound as a sixth ligand with distorted octahedral geometry at the metal; the in-plane ligand-Fe-ligand and ligand-Mn-ligand angles change by 20-30 degrees to coordinate azide as a sixth ligand. However, the positions of the distal azide nitrogens are different in the FeSOD and MnSOD complexes. The geometries of the Fe(III), Fe(II), and Fe(III)-azide species suggest a reaction mechanism for superoxide dismutation in which the metal alternates between five- and six-coordination. A reaction scheme in which the ligated solvent acts as a proton acceptor in the first half-reaction [formation of Fe(II) and oxygen] is consistent with the pH dependence of the kinetic parameters and spectroscopic properties of Fe superoxide dismutase.

X-ray analyses of wild-type and mutant flavodoxins from Clostridium beijerinckii show that the conformation of the peptide Gly57-Asp58, in a bend near the isoalloxazine ring of FMN, is correlated with the oxidation state of the FMN prosthetic group. The Gly-Asp peptide may adopt any of three conformations: trans O-up, in which the carbonyl oxygen of Gly57 (O57) points toward the flavin ring; trans O-down, in which O57 points away from the flavin; and cis O-down. Interconversions among these conformers that are linked to oxidation-reduction of the flavin can modulate the redox potentials of bound FMN. In the semiquinone and reduced forms of the protein, the Gly57-Asp58 peptide adopts the trans O-up conformation and accepts a hydrogen bond from the flavin N5H [Smith, W. W., Burnett, R. M., Darling, G. D., & Ludwig, M. L. (1977) J. Mol. Biol. 117, 195-225; Ludwig, M. L., & Luschinsky, C. L. (1992) in Chemistry and Biochemistry of Flavoenzymes III (Müller, F., Ed.) pp 427-466, CRC Press, Boca Raton, FL]. Analyses reported in this paper confirm that, in crystals of wild-type oxidized C. beijerinckii flavodoxin, the Gly57-Asp58 peptide adopts the O-down orientation and isomerizes to the cis conformation. This cis form is preferentially stabilized in the crystals by intermolecular hydrogen bonding to Asn137. Structures for the mutant Asn137Ala indicate that a mixture of all three conformers, mostly O-down, exists in oxidized C. beijerinckii flavodoxin in the absence of intermolecular hydrogen bonds. Redox potentials have been manipulated by substitutions that alter the conformational energies of the bend at 56M-G-D-E. The mutation Asp58Pro was constructed to study a case where energies for cis-trans conversion would be different from that of wild type. Intermolecular interactions with Asn137 are precluded in the crystal, yet Gly57-Pro58 is cis, and O-down, when the flavin is oxidized. Reduction of the flavin induces rearrangement to the trans O-up conformation. Redox potential shifts reflect the altered energies associated with the peptide rearrangement; E(ox/sq) decreases by approximately 60 mV (1.3 kcal/mol). Further, the results of mutation of Gly57 agree with predictions that a side chain at residue 57 should make addition of the first electron more difficult, by raising the energy of the O-up conformer that forms when the flavin is reduced to its semiquinone state. The ox/sq potentials in the mutants Gly57Ala, Gly57Asn, and Gly57Asp are all decreased by approximately 60 mV (1.3 kcal/mol). Introduction of the beta-branched threonine side chain at position 57 has much larger effects on the conformations and potentials. The Thr57-Asp58 peptide adopts a trans O-down conformation when the flavin is oxidized; upon reduction to the semiquinone, the 57-58 peptide rotates to a trans O-up conformation resembling that found in the wild-type protein. Changes in FMN-protein interactions and in conformational equilibria in G57T combine to decrease the redox potential for the ox/sq equilibrium by 180 mV (+4.0 kcal/mol) and to inc...

Phthalate dioxygenase reductase (PDR) is a prototypical iron-sulfur flavoprotein (36 kilodaltons) that utilizes flavin mononucleotide (FMN) to mediate electron transfer from the two-electron donor, reduced nicotinamide adenine nucleotide (NADH), to the one-electron acceptor, [2Fe-2S]. The crystal structure of oxidized PDR from Pseudomonas cepacia has been analyzed at 2.0 angstrom resolution resolution; reduced PDR and pyridine nucleotide complexes have been analyzed at 2.7 angstrom resolution. NADH, FMN, and the [2Fe-2S] cluster, bound to distinct domains, are brought together near a central cleft in the molecule, with only 4.9 angstroms separating the flavin 8-methyl and a cysteine sulfur ligated to iron. The domains that bind FMN and [2Fe-2S] are packed so that the flavin ring and the plane of the [2Fe-2S] core are approximately perpendicular. The [2Fe-2S] group is bound by four cysteines in a site resembling that in plant ferredoxins, but its redox potential (-174 millivolts at pH 7.0) is much higher than the potentials of plant ferredoxins. Structural and sequence similarities assign PDR to a distinct family of flavoprotein reductases, all related to ferredoxin NADP(+)-reductase.

Two X-ray structures of cobalamin (B12) bound to proteins have now been determined. These structures reveal that the B12 cofactor undergoes a major conformational change on binding to the apoenzymes of methionine synthase and methylmalonyl-coenzyme A mutase: The dimethylbenzimidazole ligand to the cobalt is displaced by a histidine residue from the protein. Two methyltransferases from archaebacteria that catalyze methylation of mercaptoethanesulfonate (coenzyme M) during methanogenesis have also been shown to contain histidine-ligated cobamides. In corrinoid iron-sulfur methyltransferases from acetogenic and methanogenic organisms, benzimidazole is dissociated from cobalt, but without replacement by histidine. Thus, dimethylbenzimidazole displacement appears to be an emerging theme in cobamide-containing methyltransferases. In methionine synthase, the best studied of the methyltransferases, the histidine ligand appears to be required for competent methyl transfer between methyl-tetrahydrofolate and homocysteine but dissociates for reductive reactivation of the inactive oxidized enzyme. Replacement of dimethylbenzimidazole by histidine may allow switching between the catalytic and activation cycles. The best-characterized B12-dependent mutases that catalyze carbon skeleton rearrangement, for which methylmalonyl-coenzyme A mutase is the prototype, also bind cobalamin cofactors with histidine as the cobalt ligand, although other cobalamin-dependent mutases do not appear to utilize histidine ligation. It is intriguing to find that mutases, which catalyze homolytic rather than heterolytic cleavage of the carbon-cobalt bond, can use this structural motif. In methylmalonylCoA mutase a significant feature, which may be important in facilitating homolytic cleavage, is the long cobalt-nitrogen bond linking histidine to the co-factor. The intermediate radical species generated in catalysis are sequestered in the relatively hydrophilic core of an alpha/beta barrel domain of the mutase.