Bound manganese oxides capable of reducing the bacteriochlorophyll dimer of modified reaction centers from Rhodobacter sphaeroides.

A biohybrid model system is described that interfaces synthetic Mn-oxides with bacterial reaction centers to gain knowledge concerning redox reactions by metal clusters in proteins, in particular the Mn4CaO5 cluster of photosystem II. The ability of Mn-oxides to bind to modified bacterial reaction centers and transfer an electron to the light-induced oxidized bacteriochlorophyll dimer, P+, was characterized using optical spectroscopy. The environment of P was altered to obtain a high P/P+ midpoint potential. In addition, different metal-binding sites were introduced by substitution of amino acid residues as well as extension of the C-terminus of the M subunit with the C-terminal region of the D1 subunit of photosystem II. The Mn-compounds MnO2, αMn2O3, Mn3O4, CaMn2O4, and Mn3(PO4)2 were tested and compared to MnCl2. In general, addition of the Mn-compounds resulted in a decrease in the amount of P+ while the reduced quinone was still present, demonstrating that the Mn-compounds can serve as secondary electron donors. The extent of P+ reduction for the Mn-oxides was largest for αMn2O3 and CaMn2O4 and smallest for Mn3O4 and MnO2. The addition of Mn3(PO4)2 resulted in nearly complete P+ reduction, similar to MnCl2. Overall, the activity was correlated with the initial oxidation state of the Mn-compound. Transient optical measurements showed a fast kinetic component, assigned to reduction of P+ by the Mn-oxide, in addition to a slow component due to charge recombination. The results support the conjecture that the incorporation of Mn-oxides by ancient anoxygenic phototrophs was a step in the evolution of oxygenic photosynthesis.

Influence of the Electrochemical Properties of the Bacteriochlorophyll Dimer on Triplet Energy-Transfer Dynamics in Bacterial Reaction Centers.

Energetics, protein dynamics, and electronic coupling are the key factors in controlling both electron and energy transfer in photosynthetic bacterial reaction centers (RCs). Here, we examine the rates and mechanistic pathways of the P+HA- radical-pair charge recombination, triplet state formation, and subsequent triplet energy transfer from the triplet state of the bacteriochlorophyll dimer (P) to the carotenoid in a series of mutant RCs (L131LH + M160LH (D1), L131LH + M197FH (D2), and L131LH + M160LH + M197FH (T1)) of Rhodobacter sphaeroides. In these mutants, the electronic structure of P is perturbed and the P/P+ midpoint potential is systematically increased due to addition of hydrogen bonds between P and the introduced residues. High-resolution, broad-band, transient absorption spectroscopy on the femtosecond to microsecond timescale shows that the charge recombination rate increases and the triplet energy transfer rate decreases in these mutants relative to the wild type (WT). The increase of the charge recombination rate is correlated to the increase in the energy level of P+HA- and the increase in the P/P+ midpoint potential. On the other hand, the decrease in rate of triplet energy transfer in the mutants can be explained in terms of a lower energy of 3P and a shift in the electron spin density distribution in the bacteriochlorophylls of P. The triplet energy-transfer rate follows the order of WT > L131LH + M197FH > L131LH + M160LH > L131LH + M160LH + M197FH, both at room temperature and at 77 K. A pronounced temperature dependence of the rate is observed for all of the RC samples. The activation energy associated to this process is increased in the mutants relative to WT, consistent with a lower 3P energy due to the addition of hydrogen bonds between P and the introduced residues.

Binding and Energetics of Electron Transfer between an Artificial Four-Helix Mn-Protein and Reaction Centers from Rhodobacter sphaeroides.

The ability of an artificial four-helix bundle Mn-protein, P1, to bind and transfer an electron to photosynthetic reaction centers from the purple bacterium Rhodobacter sphaeroides was characterized using optical spectroscopy. Upon illumination of reaction centers, an electron is transferred from P, the bacteriochlorophyll dimer, to QA, the primary electron acceptor. The P1 Mn-protein can bind to the reaction center and reduce the oxidized bacteriochlorophyll dimer, P+, with a dissociation constant of 1.2 µM at pH 9.4, comparable to the binding constant of c-type cytochromes. Amino acid substitutions of surface residues on the Mn-protein resulted in increases in the dissociation constant to 8.3 µM. The extent of reduction of P+ by the P1 Mn-protein was dependent on the P/P+ midpoint potential and the pH. Analysis of the free energy difference yielded a midpoint potential of approximately 635 mV at pH 9.4 for the Mn cofactor of the P1 Mn-protein, a value similar to those found for other Mn cofactors in proteins. The linear dependence of -56 mV/pH is consistent with one proton being released upon Mn oxidation, allowing the complex to maintain overall charge neutrality. These outcomes demonstrate the feasibility of designing four-helix bundles and other artificial metalloproteins to bind and transfer electrons to bacterial reaction centers and establish the usefulness of this system as a platform for designing sites to bind novel metal cofactors capable of performing complex oxidation-reduction reactions.

Biochemical and spectroscopic characterization of dinuclear Mn-sites in artificial four-helix bundle proteins.

To better understand metalloproteins with Mn-clusters, we have designed artificial four-helix bundles to have one, two, or three dinuclear metal centers able to bind Mn(II). Circular dichroism measurements showed that the Mn-proteins have substantial α-helix content, and analysis of electron paramagnetic resonance spectra is consistent with the designed number of bound Mn-clusters. The Mn-proteins were shown to catalyze the conversion of hydrogen peroxide into molecular oxygen. The loss of hydrogen peroxide was dependent upon the concentration of protein with bound Mn, with the proteins containing multiple Mn-clusters showing greater activity. Using an oxygen sensor, the oxygen concentration was found to increase with a rate up to 0.4µM/min, which was dependent upon the concentrations of hydrogen peroxide and the Mn-protein. In addition, the Mn-proteins were shown to serve as electron donors to bacterial reaction centers using optical spectroscopy. Similar binding of the Mn-proteins to reaction centers was observed with an average dissociation constant of 2.3µM. The Mn-proteins with three metal centers were more effective at this electron transfer reaction than the Mn-proteins with one or two metal centers. Thus, multiple Mn-clusters can be incorporated into four-helix bundles with the capability of performing catalysis and electron transfer to a natural protein.

Design of dinuclear manganese cofactors for bacterial reaction centers.

A compelling target for the design of electron transfer proteins with novel cofactors is to create a model for the oxygen-evolving complex, a Mn4Ca cluster, of photosystem II. A mononuclear Mn cofactor can be added to the bacterial reaction center, but the addition of multiple metal centers is constrained by the native protein architecture. Alternatively, metal centers can be incorporated into artificial proteins. Designs for the addition of dinuclear metal centers to four-helix bundles resulted in three artificial proteins with ligands for one, two, or three dinuclear metal centers able to bind Mn. The three-dimensional structure determined by X-ray crystallography of one of the Mn-proteins confirmed the design features and revealed details concerning coordination of the Mn center. Electron transfer between these artificial Mn-proteins and bacterial reaction centers was investigated using optical spectroscopy. After formation of a light-induced, charge-separated state, the experiments showed that the Mn-proteins can donate an electron to the oxidized bacteriochlorophyll dimer of modified reaction centers, with the Mn-proteins having additional metal centers being more effective at this electron transfer reaction. Modeling of the structure of the Mn-protein docked to the reaction center showed that the artificial protein likely binds on the periplasmic surface similarly to cytochrome c2, the natural secondary donor. Combining reaction centers with exogenous artificial proteins provides the opportunity to create ligands and investigate the influence of inhomogeneous protein environments on multinuclear redox-active metal centers. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Conversion of aminodeoxychorismate synthase into anthranilate synthase with Janus mutations: mechanism of pyruvate elimination catalyzed by chorismate enzymes.

The central importance of chorismate enzymes in bacteria, fungi, parasites, and plants combined with their absence in mammals makes them attractive targets for antimicrobials and herbicides. Two of these enzymes, anthranilate synthase (AS) and aminodeoxychorismate synthase (ADCS), are structurally and mechanistically similar. The first catalytic step, amination at C2, is common between them, but AS additionally catalyzes pyruvate elimination, aromatizing the aminated intermediate to anthranilate. Despite prior attempts, the conversion of a pyruvate elimination-deficient enzyme into an elimination-proficient one has not been reported. Janus, a bioinformatics method for predicting mutations required to functionally interconvert homologous enzymes, was employed to predict mutations to convert ADCS into AS. A genetic selection on a library of Janus-predicted mutations was performed. Complementation of an AS-deficient strain of Escherichia coli grown on minimal medium led to several ADCS mutants that allow growth in 6 days compared to 2 days for wild-type AS. The purified mutant enzymes catalyze the conversion of chorismate to anthranilate at rates that are ∼50% of the rate of wild-type ADCS-catalyzed conversion of chorismate to aminodeoxychorismate. The residues mutated do not contact the substrate. Molecular dynamics studies suggest that pyruvate elimination is controlled by the conformation of the C2-aminated intermediate. Enzymes that catalyze elimination favor the equatorial conformation, which presents the C2-H to a conserved active site lysine (Lys424) for deprotonation and maximizes stereoelectronic activation. Acid/base catalysis of pyruvate elimination was confirmed in AS and salicylate synthase by showing incorporation of a solvent-derived proton into the pyruvate methyl group and by solvent kinetic isotope effects on pyruvate elimination catalyzed by AS.