Keinan, E.

J. Am. Chem. Soc. 1999, 121, 8978-8982, 10.1021/ja990314q

An antibody−metalloporphyrin assembly that catalyzes the enantioselective oxidation of aromatic sulfides to sulfoxides is presented. Antibody SN37.4 was elicited against a water-soluble tin(IV) porphyrin containing an axial α-naphthoxy ligand. The catalytic assembly comprising antibody SN37.4 and a ruthenium(II) porphyrin cofactor exhibited typical enzyme characteristics, such as predetermined oxidant and substrate selectivity, enantioselective delivery of oxygen to the substrate, and Michaelis−Menten saturation kinetics. This assembly, which promotes a complex, multistep catalytic event, represents a close model of natural heme-dependent oxidation enzymes.

Schultz, P.G.

J. Am. Chem. Soc. 2008, 130, 13194-13195, 10.1021/ja804653f

An E. coli catabolite activator protein (CAP) has been converted into a sequence-specific DNA cleaving protein by genetically introducing (2,2′-bipyridin-5-yl)alanine (Bpy-Ala) into the protein. The mutant CAP (CAP-K26Bpy-Ala) showed comparable binding affinity to CAP-WT for the consensus operator sequence. In the presence of Cu(II) and 3-mercaptopropionic acid, CAP-K26Bpy-Ala cleaves double-stranded DNA with high sequence specificity. This method should provide a useful tool for mapping the molecular details of protein−nucleic acid interactions.

Ghirlanda, G.

Biopolymers 2013, 100, 558-571, 10.1002/bip.22420

Over the last 25 years, de novo design has proven to be a valid approach to generate novel, well‐folded proteins, and most recently, functional proteins. In response to societal needs, this approach is been used increasingly to design functional proteins developed with an eye toward sustainable fuel production. This review surveys recent examples of bioinspired de novo designed peptide based catalysts, focusing in particular on artificial hydrogenases.

Ghirlanda, G.

Methods Enzymol. 2016, 389-416, 10.1016/bs.mie.2016.06.001

The last decades have seen an increased interest in finding alternative means to produce renewable fuels in order to satisfy the growing energy demands and to minimize environmental impact. Nature can serve as an inspiration for development of these methodologies, as enzymes are able to carry out a wide variety of redox processes at high efficiency, employing a wide array of earth-abundant transition metals to do so. While it is well recognized that the protein environment plays an important role in tuning the properties of the different metal centers, the structure/function relationships between amino acids and catalytic centers are not well resolved. One specific approach to study the role of proteins in both electron and proton transfer is the biomimetic design of redox active peptides, binding organometallic clusters in well-understood protein environments. Here we discuss different strategies for the design of peptides incorporating redox active FeS clusters, [FeFe]-hydrogenase organometallic mimics, and porphyrin centers into different peptide and protein environments in order to understand natural redox enzymes.

Schultz, P.G.

Science 1987, 238, 1401-1403, 10.1126/science.3685986

The relatively nonspecific single-stranded deoxyribonuclease, staphylococcal nuclease, was selectively fused to an oligonucleotide binding site of defined sequence to generate a hybrid enzyme. A cysteine was substituted for Lys116 in the enzyme by oligonucleotide-directed mutagenesis and coupled to an oligonucleotide that contained a 3'-thiol. The resulting hybrid enzyme cleaved single-stranded DNA at sites adjacent to the oligonucleotide binding site.

Kazlauskas, R.J.

Chem. - Eur. J. 2006, 12, 1587-1596, 10.1002/chem.200501413

Carbonic anhydrase is a zinc metalloenzyme that catalyzes the hydration of carbon dioxide to bicarbonate. Replacing the active‐site zinc with manganese yielded manganese‐substituted carbonic anhydrase (CA[Mn]), which shows peroxidase activity with a bicarbonate‐dependent mechanism. In the presence of bicarbonate and hydrogen peroxide, (CA[Mn]) catalyzed the efficient oxidation of o‐dianisidine with kcat/KM=1.4×106 m−1 s−1, which is comparable to that for horseradish peroxidase, kcat/KM=57×106 m−1 s−1. CA[Mn] also catalyzed the moderately enantioselective epoxidation of olefins to epoxides (E=5 for p‐chlorostyrene) in the presence of an amino‐alcohol buffer, such as N,N‐bis(2‐hydroxyethyl)‐2‐aminoethanesulfonic acid (BES). This enantioselectivity is similar to that for natural heme‐based peroxidases, but has the advantage that CA[Mn] avoids the formation of aldehyde side products. CA[Mn] degrades during the epoxidation limiting the yield of the epoxidations to <12 %. Replacement of active‐site residues Asn62, His64, Asn67, Gln92, or Thr200 with alanine by site‐directed mutagenesis decreased the enantioselectivity demonstrating that the active site controls the enantioselectivity of the epoxidation.

Kazlauskas, R.J.

Top. Organomet. Chem. 2009, 10.1007/3418_2008_1

Carbonic anhydrase binds a zinc ion in a hydrophobic active site using the imidazole groups of three histidine residues. The natural role of carbonic anhydrase is to catalyze the reversible hydration of carbon dioxide to bicarbonate, but it also catalyzes hydrolysis of esters with moderate enantioselectivity. Replacing the active-site zinc with manganese yielded manganese-substituted carbonic anhydrase (CA[Mn]), which shows peroxidase activity with a bicarbonate-dependent mechanism. In the presence of bicarbonate and hydrogen peroxide, CA[Mn] catalyzed the efficient oxidation of o-dianisidine with k cat /K M = 1.4 × 106 M−1s−1, which is comparable to that for horseradish peroxidase, k cat /K M = 57 × 106 M−1s−1. CA[Mn] also catalyzed the moderately enantioselective epoxidation of olefins to epoxides (E = 5 for p-chlorostyrene). This enantioselectivity is similar to that for natural heme-based peroxidases, but has the advantage that CA[Mn] avoids formation of aldehyde side products. CA[Mn] degrades during the epoxidation, limiting the yield of the epoxidations to <12%. Replacement of active-site residues Asn62, His64, Asn67, Gln92, or Thr200 with alanine by site-directed mutagenesis decreased the enantioselectivity showing that the active site controls enantioselectivity of the epoxidation.

Schultz, P.G.

J. Am. Chem. Soc. 1990, 112, 9414-9415, 10.1021/ja00181a065

The specificity and diversity of the immune system have recently been exploited in the generation of antibodies that catalyze a wide variety of chemical reactions.1·2 Several general strategies for the design of catalytic antibodies have emerged, including the use of antibody binding energy to enhance the chemical reactivity of a cofactor or to position a cofactor and a substrate in close proximity.3,4 An intriguing target for antibody-cofactor catalysis is the oxidative reactions characteristic of heme proteins. Here we report that antibodies specific for A-methylmesoporphyrin IX bind iron(III) mesoporphyrin IX and that the resulting complex catalyzes the oxidation of several substrates. These studies are a first step toward the development of selective antibody-heme monooxygenase catalysts.

Ghirlanda, G.

Chem. Commun. 2014, 50, 15852-15855, 10.1039/c4cc06700b

Hydrogen is an attractive fuel with potential for production scalability, provided that inexpensive, efficient molecular catalysts utilizing base metals can be developed for hydrogen production. Here we show for the first time that cobalt myoglobin (CoMyo) catalyzes hydrogen production in mild aerobic conditions with turnover number of 520 over 8 hours. Compared to free Co-protoporphyrin IX, incorporation into the myoglobin scaffold results in a 4-fold increase in photoinduced hydrogen production activity. Engineered variants in which specific histidine resides in proximity of the active site were mutated to alanine result in modulation of the catalytic activity, with the H64A/H97A mutant displaying activity 2.5-fold higher than wild type. Our results demonstrate that protein scaffolds can augment and modulate the intrinsic catalytic activity of molecular hydrogen production catalysts.

Ghirlanda, G.

Biochim. Biophys. Acta, Bioenerg. 2016, 1857, 598-603, 10.1016/j.bbabio.2015.09.001

Bioinspired, protein-based molecular catalysts utilizing base metals at the active are emerging as a promising avenue to sustainable hydrogen production. The protein matrix modulates the intrinsic reactivity of organometallic active sites by tuning second-sphere and long-range interactions. Here, we show that swapping Co-Protoporphyrin IX for Fe-Protoporphyrin IX in cytochrome b562 results in an efficient catalyst for photoinduced proton reduction to molecular hydrogen. Further, the activity of wild type Co-cyt b562 can be modulated by a factor of 2.5 by exchanging the coordinating methionine with alanine or aspartic acid. The observed turnover numbers (TON) range between 125 and 305, and correlate well with the redox potential of the Co-cyt b562 mutants. The photosensitized system catalyzes proton reduction with high efficiency even under an aerobic atmosphere, implicating its use for biotechnological applications. 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.

Kazlauskas, R.J.

ChemCatChem 2010, 2, 953-957, 10.1002/cctc.201000159

CA confidential: Replacing the active‐site zinc in carbonic anhydrase (CA) by rhodium forms a new enzymatic catalyst for cofactor‐free hydroformylation of styrene with syn gas. Unlike free rhodium, this rhodium–protein hybrid, [Rh]–CA, is regioselective (8.4:1) for linear over branched aldehyde product, which is a 40‐fold change in regioselectivity compared to free rhodium.

Kazlauskas, R.J.

Chem. - Eur. J. 2009, 15, 1370-1376, 10.1002/chem.200801673

One useful synthetic reaction missing from nature's toolbox is the direct hydrogenation of substrates using hydrogen. Instead nature uses cofactors like NADH to reduce organic substrates, which adds complexity and cost to these reductions. To create an enzyme that can directly reduce organic substrates with hydrogen, researchers have combined metal hydrogenation catalysts with proteins. One approach is an indirect link where a ligand is linked to a protein and the metal binds to the ligand. Another approach is direct linking of the metal to protein, but nonspecific binding of the metal limits this approach. Herein, we report a direct hydrogenation of olefins catalyzed by rhodium(I) bound to carbonic anhydrase (CA‐[Rh]). We minimized nonspecific binding of rhodium by replacing histidine residues on the protein surface using site‐directed mutagenesis or by chemically modifying the histidine residues. Hydrogenation catalyzed by CA‐[Rh] is slightly slower than for uncomplexed rhodium(I), but the protein environment induces stereoselectivity favoring cis‐ over trans‐stilbene by about 20:1. This enzyme is the first cofactor‐independent reductase that reduces organic molecules using hydrogen. This catalyst is a good starting point to create variants with tailored reactivity and selectivity. This strategy to insert transition metals in the active site of metalloenzymes opens opportunities to a wider range of enzyme‐catalyzed reactions.

Keinan, E.

Pure Appl. Chem. 1990, 62, 2013-2019, 10.1351/pac199062102013

An attempt was made to mimic cytochrome P-450-like activity using antibodies elicited against metallo-porphyrins. Monoclonal antibodies raised against a water-soluble Sn(1V) porphyrin complex (1) exhibited Specificity for a variety of monomeric metalloporphyrins, as well as for the b-0x0-Fe(III) porphyrin dimer 2. Some antibodies were found to be more selective for the monomer 1 than for the dimer 2, suggesting an "edge-on" recognition of the planar porphyrin molecule. The catalytic activity of the antibody-metalloporphyrin complexes was investigated using the epoxidation of styrene by iodosobenzene as a model reaction. Three biphasic media were studied for this reaction: reverse micelles, microemulsions, and solid catalyst in organic solvent. The most promising results were obtained with solid catalyst (obtained via lyophilization of equimolar amounts of Mn(TCP)Cl and specific antibody) in dry CHzClz at room temperature, as indicated by the high turnover numbers of the catalyst. A difference in the relative activity of the various monoclonal antibodies (MABs) was noted. The anti-1 antibodies displayed ca. 30-60% higher activity compared to a nonrelevant MAB.