FEBS Letters

doi: 10.1002/1873-3468.14379pmid: N/A

Högbom, Martin

doi: 10.1002/1873-3468.14559pmid: 36623847

Lubitz, Wolfgang; Pantazis, Dimitrios A.; Cox, Nicholas

doi: 10.1002/1873-3468.14543pmid: 36409002

The understanding of light‐induced biological water oxidation in oxygenic photosynthesis is of great importance both for biology and (bio)technological applications. The chemically difficult multistep reaction takes place at a unique protein‐bound tetra‐manganese/calcium cluster in photosystem II whose structure has been elucidated by X‐ray crystallography (Umena et al. Nature 2011, 473, 55). The cluster moves through several intermediate states in the catalytic cycle. A detailed understanding of these intermediates requires information about the spatial and electronic structure of the Mn4Ca complex; the latter is only available from spectroscopic techniques. Here, the important role of Electron Paramagnetic Resonance (EPR) and related double resonance techniques (ENDOR, EDNMR), complemented by quantum chemical calculations, is described. This has led to the elucidation of the cluster's redox and protonation states, the valence and spin states of the manganese ions and the interactions between them, and contributed substantially to the understanding of the role of the protein surrounding, as well as the binding and processing of the substrate water molecules, the O‐O bond formation and dioxygen release. Based on these data, models for the water oxidation cycle are developed.

Simon, Philipp S.; Makita, Hiroki; Bogacz, Isabel; Fuller, Franklin; Bhowmick, Asmit; Hussein, Rana; Ibrahim, Mohamed; Zhang, Miao; Chatterjee, Ruchira; Cheah, Mun Hon; Chernev, Petko; Doyle, Margaret D.; Brewster, Aaron S.; Alonso‐Mori, Roberto; Sauter, Nicholas K.;

Siegbahn, Per E. M.

doi: 10.1002/1873-3468.14512pmid: 36254111

A computational methodology is briefly described, which appears to be able to accurately describe the mechanisms of redox active enzymes. The method is built on hybrid density functional theory where the inclusion of a fraction of exact exchange is critical. Two examples of where the methodology has been applied are described. The first example is the mechanism for water oxidation in photosystem II, and the second one is the mechanism for N2 activation by nitrogenase. The mechanism for PSII has obtained very strong support from subsequent experiments. For nitrogenase, the calculations suggest that there should be an activation process prior to catalysis, which is still strongly debated.

Threatt, Stephanie D.; Rees, Douglas C.

doi: 10.1002/1873-3468.14534pmid: 36344435

Nitrogenase is the sole enzyme responsible for the ATP‐dependent conversion of atmospheric dinitrogen into the bioavailable form of ammonia (NH3), making this protein essential for the maintenance of the nitrogen cycle and thus life itself. Despite the widespread use of the Haber–Bosch process to industrially produce NH3, biological nitrogen fixation still accounts for half of the bioavailable nitrogen on Earth. An important feature of nitrogenase is that it operates under physiological conditions, where the equilibrium strongly favours ammonia production. This biological, multielectron reduction is a complex catalytic reaction that has perplexed scientists for decades. In this review, we explore the current understanding of the molybdenum nitrogenase system based on experimental and computational research, as well as the limitations of the crystallographic, spectroscopic, and computational techniques employed. Finally, essential outstanding questions regarding the nitrogenase system will be highlighted alongside suggestions for future experimental and computational work to elucidate this essential yet elusive process.

Ravanfar, Raheleh; Sheng, Yuling; Gray, Harry B.; Winkler, Jay R.

doi: 10.1002/1873-3468.14514pmid: 36250256

Flavocytochrome P450 from Bacillus megaterium (P450BM3) is a natural fusion protein containing reductase and heme domains. In the presence of NADPH and dioxygen the enzyme catalyses the hydroxylation of long‐chain fatty acids. Analysis of the P450BM3 structure reveals chains of closely spaced tryptophan and tyrosine residues that might serve as pathways for high‐potential oxidizing equivalents to escape from the heme active site when substrate oxidation is not possible. Our investigations of the total number of enzyme turnovers before deactivation have revealed that replacement of selected tryptophan and tyrosine residues with redox inactive groups leads to a twofold reduction in enzyme survival time. Tryptophan‐96 is critical for prolonging enzyme activity, suggesting a key protective role for this residue.

Kipouros, Ioannis; Solomon, Edward I.

doi: 10.1002/1873-3468.14503pmid: 36178078

Tyrosinase is the most predominant member of the coupled binuclear copper (CBC) protein family. The recent trapping and spectroscopic definition of the elusive catalytic ternary intermediate (enzyme/O2/monophenol) of tyrosinase dictates a monooxygenation mechanism that revises previous proposals and involves cleavage of the μ‐η2:η2‐peroxide dicopper(II) O–O bond to accept the phenolic proton, followed by monophenolate coordination to copper concomitant with aromatic hydroxylation by the non‐protonated μ‐oxo. Here, we compare and contrast previously proposed and current mechanistic models for monophenol monooxygenation of tyrosinase. Next, we discuss how these recent insights provide new opportunities towards uncovering structure–function relationships in CBC enzymes, as well as understanding fundamental principles for O2 activation and reactivity by bioinorganic active sites.

Klinman, Judith P.

doi: 10.1002/1873-3468.14515pmid: 36239559

Formulations of hydrogen tunneling in enzyme‐catalysed C–H activation reactions indicate enthalpic barriers to reaction that are independent of chemical steps and dependent on the protein scaffold. A tool to identify catalytically relevant site‐specific protein thermal networks has emerged from temperature‐dependent hydrogen deuterium exchange (TDHDX). Focusing on mutant enzyme forms with altered activation energies for catalysis, TDHDX provides a comparative analysis of the impact of mutation on Ea for local protein unfolding. Identified thermal networks appear unrelated to protein scaffold conservation and track to the dictates of the catalysed reaction, including sites for metal binding. The positions of thermal networks provide a framework for further understanding of time‐dependent, functionally relevant protein motions. Measurement of nanosecond Stokes shifts at the surface of the thermal network in soybean lipoxygenase yields activation energies that are identical to Ea values measured for kcat. This finding identifies a rapid (> nanosecond), long‐range and cooperative structural reorganization as the thermal barrier to catalysis. A model for protein dynamics is put forward that integrates broadly distributed protein conformational sampling with protein embedded thermal networks.

Broderick, Joan B.; Broderick, William E.; Hoffman, Brian M.

doi: 10.1002/1873-3468.14519pmid: 36251330

Enzymes that use a [4Fe‐4S]1+ cluster plus S‐adenosyl‐l‐methionine (SAM) to initiate radical reactions (radical SAM) form the largest enzyme superfamily, with over half a million members across the tree of life. This review summarizes recent work revealing the radical SAM reaction pathway, which ultimately liberates the 5′‐deoxyadenosyl (5′‐dAdo•) radical to perform extremely diverse, highly regio‐ and stereo‐specific, transformations. Most surprising was the discovery of an organometallic intermediate Ω exhibiting an Fe‐C5′‐adenosyl bond. Ω liberates 5′‐dAdo• through homolysis of the Fe–C5′ bond, in analogy to Co–C5′ bond homolysis in B12, previously viewed as biology's paradigmatic radical generator. The 5′‐dAdo• has been trapped and characterized in radical SAM enzymes via a recently discovered photoreactivity of the [4Fe‐4S]+/SAM complex, and has been confirmed as a catalytically active intermediate in enzyme catalysis. The regioselective SAM S–C bond cleavage to produce 5′‐dAdo• originates in the Jahn–Teller effect. The simplicity of SAM as a radical precursor, and the exquisite control of 5′‐dAdo• reactivity in radical SAM enzymes, may be why radical SAM enzymes pervade the tree of life, while B12 enzymes are only a few.