Paul W. King

Polymer-bound nanodiscs, such as styrene maleic acid lipid particles (SMALPs), have emerged as a technique for isolating membrane proteins that retain an annulus of native lipids, in bilayer conformation, surrounding the encapsulated protein. The preservation of the native membrane environment in these so-called “native nanodiscs” has been reported to exhibit more native-like biophysical activity in vitro for various protein complexes, across a myriad of biophysical studies, typically conducted free in solution. Here, we combine the SMALP technique with the Langmuir–Blodgett trough, to produce thin films comprised of photosystem I (PSI) SMALPs. This approach presents the ability to reconstitute of the native membrane architecture of uniform PSI arrays as seen in nature. Here, we report the conditions required to partition PSI–SMALPs to the air–water interface and achieve controlled compression into thin films. These parameters concern the physical interaction of SMALP particles and may be transferrable to other protein and lipid only SMALP systems. Furthermore, PSI–SMALP thin films exhibit greater stability than those comprised of PSI detergent micelles and demonstrate a lower surface compression modulus compared to lipid only monolayers. Individual PSI–SMALPs retain their size following decompression from the thin film conformation, consistent with retention of the overall PSI–SMALP particle architecture. This platform offers a more accurate biomimetic system from which to probe fundamental questions and drive applied work.

Ferredoxins are central to cellular metabolism by mediating electron flow in energy conversion reactions. The focus of this study was to systematically examine twelve ferredoxin and ferredoxin‐like proteins from Synechocystis sp. PCC 6803 to identify their properties, activities, and functions in electron transfer. Using electron paramagnetic resonance spectroscopy, we detected cluster types consistent with major ferredoxin families including plant‐type [2Fe‐2S], adrenodoxin, thioredoxin, and bacterial‐type [4Fe‐4S] ferredoxins. In addition, we found that the ssr3184 ferredoxin‐like protein exchanged between a [3Fe‐4S] or a [4Fe‐4S] cluster, pointing to a possible functional change in response to changes in oxygen or cellular redox poise. Electrochemical measurements demonstrated that these ferredoxins constitute a broad potential window, from −243 mV to −520 mV vs SHE. Investigations on their capacity to support electron transfer focused on reactions with two major redox hubs: Photosystem I and pyruvate:ferredoxin oxidoreductase and included testing of binding interactions with nitrite reductase. Expression profiling under multiple environmental conditions was also used to predict function and revealed distinct regulatory patterns. Collectively, these findings identified a group of core ferredoxins that directly support photosynthetic electron transfer, and more specialized ones that may serve other functions. In summary, Synechocystis utilizes a suite of ferredoxins to maintain cellular redox homeostasis under dynamic environmental conditions.

Clostridium pasteurianum [FeFe]-hydrogenase II (CpII) is distinguished by having a ~103-fold preference for catalyzing the H2 oxidation reaction over H2 production. Here we used electron paramagnetic resonance and infrared spectroscopy to identify catalytic intermediates and role of the accessory [4Fe-4S] clusters in electron transport. In the resting state, CpII consisted of an oxidized, Hox, H-cluster and diamagnetic accessory F-clusters. Under reduction with H2, CpII converted to a singly reduced H-cluster, or Hred, whereas chemical reduction led to a mixture of Hred and the two-electron, super-reduced, Hsred, state. Exchange of a conserved proton-transfer cysteine for serine led to increased levels of Hsred. Analysis of the magnetic properties of reduced CpII revealed extensive spin-coupling among the F-clusters, and between F-clusters and the Hsred H-cluster state. We propose that the H2 oxidation bias in CpII is a synergistic outcome of the H-cluster electronic structure and spin-coupling to favor H2 binding and oxidation.

Photochemical reduction reactions catalyzed by nanocrystals (NCs) and enzymes require both electron transfer (ET) and hole transfer (HT) reactions to occur in spatial and temporal proximity. As a result, the observed photochemical reaction rates are complex functions of many competing ET and HT reactions. To facilitate interpretation of complex photochemical datasets, we developed a model that simultaneously evaluates the rates of both the reduction and oxidation reactions. We applied this model to electron-intensive reduction of dinitrogen to ammonia by CdS NCs and nitrogenase MoFe protein. A fit of the model to the kinetics of product accumulation determined that during prolonged illuminations the efficiency of N2 reduction was limited by MoFe protein oxidation, facilitated by the accumulation of the products of the oxidation reaction. Modeling time-resolved populations of catalytic intermediates revealed variability in the rates of reduction, indicating that accumulation of hydrides and N2 activation influenced the rates of electron transfer.

Ferredoxins are central to cellular metabolism by mediating electron flow in energy conversion reactions. The focus of this study was to systematically examine twelve ferredoxin and ferredoxin-like proteins from Synechocystis sp. PCC 6803 to identify their properties, activities, and functions in electron transfer. Using electron paramagnetic resonance spectroscopy, we detected cluster types consistent with major ferredoxin families including plant-type [2Fe-2S], adrenodoxin, thioredoxin, and bacterial-type [4Fe– 4S] ferredoxins. In addition, we found that the ssr3184 ferredoxin-like protein exchanged between a [3Fe–4S] or a [4Fe–4S] cluster, pointing to a possible functional change in response to changes in oxygen or cellular redox poise. Electrochemical measurements demonstrated that these ferredoxins constitute a broad potential window, from −243 mV to −520 mV vs SHE. Investigations on their capacity to support electron-transfer focused on reactions with two major redox hubs: Photosystem I and pyruvate:ferredoxin oxidoreductase and included testing of binding interactions with nitrite reductase. Expression profiling under multiple environmental conditions was also used to predict function and revealed distinct regulatory patterns. Collectively, these findings identified a group of core ferredoxins that directly support photosynthetic electron transfer, and more specialized ones that may serve other functions. In summary, Synechocystis utilizes a suite of ferredoxins to maintain cellular redox homeostasis under dynamic environmental conditions.

Developing systems that can efficiently capture photon energy and convert this energy into fuels and chemicals requires understanding how to assemble molecular components with diverse functions into complete systems possessing selectivity and efficiency in directing charge carriers to catalytic reactions. There are many challenges to achieving this goal. One promising approach is the development of hybrid systems that combine semiconductor nanocrystals (NCs) for light capture and enzymes as efficient catalysts. Such biohybrid systems capitalize on the tunable electronic and optical properties of NCs while leveraging the unmatched specificity and efficiency of enzymes in catalyzing chemical reactions, thereby offering opportunities to surpass the limitations of each component alone. Here, we focus on recent progress in developing a biohybrid system that combines CdS NCs for photon capture with the enzyme nitrogenase to accomplish light-driven dinitrogen (N₂) reduction to ammonia (NH₃). Integrating light-harvesting materials with biological catalysts requires a deep understanding of NC properties, protein stability, and electron transfer (ET), making it an inherently multidisciplinary problem. The reduction of N₂ to NH₃ is a challenging reaction, with a high demand in both agriculture and industrial chemical production. This reaction is intrinsically energy intensive, due to the need to activate the N≡N triple bond. The current standard industrial approach to N₂ reduction, the Haber–Bosch reaction, obtains the necessary energy input from fossil fuels, whereas biological systems capable of N₂ reduction utilize the hydrolysis of ATP as their energy source. Replacing these costly, energy-intensive inputs with renewable light energy represents a critical step toward sustainable NH₃ production. Recent progress has demonstrated that semiconductor CdS NCs can be coupled to the catalytic component of nitrogenase, the MoFe protein, to form a biohybrid CdS NC:MoFe protein complex, enabling light-driven N₂ reduction rather than energy input from fossil fuels or ATP. This illustrates how inorganic NCs can functionally replace the natural Fe protein partner, yielding a biohybrid catalyst that enables controlled electron delivery and provides not only light-driven NH₃ production but also new approaches for probing enzyme catalytic function. The CdS NC:MoFe protein biohybrid system enables light-initiated electron delivery at ambient temperature, as well as temperatures below freezing, allowing for stabilization and spectroscopic characterization of key reaction intermediates. These findings highlight how photochemical biohybrids can serve as both functional catalysts and mechanistic probes. Beyond studies of the nitrogenase mechanism, studies of the CdS NC:MoFe system reveal how variables such as NC size, electrostatic binding interactions, and sacrificial electron donors (SEDs) govern complex stability, charge transfer efficiency, and catalytic performance. In addition, studies of nitrogenase and the high activation barrier for N₂ reduction are enabling investigations of new and interesting questions regarding the properties and limitations of NC biocatalysis. In this Account, we describe the key features of CdS NC:MoFe protein biohybrids and the parameters for optimal light-driven N₂ reduction, and how controlling ET with light illuminates the path to new insights into the nitrogenase mechanism.