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Photoinduced charge-transfer dynamics and the influence of cluster size on the dynamics were investigated using five iron–sulfur clusters: the 1Fe–4S cluster in Pyrococcus furiosusrubredoxin, the 2Fe–2S cluster in Pseudomonas putidaputidaredoxin, the 4Fe–4S cluster in nitrogenase iron protein, and the 8Fe–7S P-cluster and the 7Fe–9S–1Mo FeMo cofactor in nitrogenase MoFe protein. Laser excitation promotes the iron–sulfur clusters to excited electronic states that relax to lower states. The electronic relaxation lifetimes of the 1Fe–4S, 8Fe–7S, and 7Fe–9S–1Mo clusters are on the picosecond time scale, although the dynamics of the MoFe protein is a mixture of the dynamics of the latter two clusters. The lifetimes of the 2Fe–2S and 4Fe–4S clusters, however, extend to several nanoseconds. A competition between reorganization energies and the density of electronic states (thus electronic coupling between states) mediates the charge-transfer lifetimes, with the 2Fe–2S cluster of Pdx and the 4Fe–4S cluster of Fe protein lying at the optimum leading to them having significantly longer lifetimes. Their long lifetimes make them the optimal candidates for long-range electron transfer and as external photosensitizers for other photoactivated chemical reactions like solar hydrogen production. Potential electron-transfer and hole-transfer pathways that possibly facilitate these charge transfers are proposed.

We used a novel experimental setup to conduct the first synchrotron-based 61Ni Mössbauer spectroscopy measurements in the energy domain on Ni coordination complexes and metalloproteins. A representative set of samples was chosen to demonstrate the potential of this approach. 61NiCr2O4was examined as a case with strong Zeeman splittings. Simulations of the spectra yielded an internal magnetic field of 44.6 T, consistent with previous work by the traditional 61Ni Mössbauer approach with a radioactive source. A linear Ni amido complex, 61Ni{N(SiMe3)Dipp}2, where Dipp = C6H3-2,6-iPr2, was chosen as a sample with an “extreme” geometry and large quadrupole splitting. Finally, to demonstrate the feasibility of metalloprotein studies using synchrotron-based 61Ni Mössbauer spectroscopy, we examined the spectra of 61Ni-substituted rubredoxin in reduced and oxidized forms, along with [Et4N]2[61Ni(SPh)4] as a model compound. For each of the above samples, a reasonable spectrum could be obtained in ∼1 d. Given that there is still room for considerable improvement in experimental sensitivity, synchrotron-based 61Ni Mössbauer spectroscopy appears to be a promising alternative to measurements with radioactive sources.

Virtually all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes, the compositions of which are largely undefined. They cannot be predicted solely from bioinformatics analyses nor are there well defined techniques currently available to unequivocally identify protein complexes (PCs). To address this issue, we attempted to directly determine the identity of PCs from native microbial biomass using Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100 °C, as the model organism. Novel PCs were identified by large scale fractionation of the native proteome using non-denaturing, sequential column chromatography under anaerobic, reducing conditions. A total of 967 distinct P. furiosus proteins were identified by mass spectrometry (nano LC-ESI-MS/MS), representing ∼80% of the cytoplasmic proteins. Based on the co-fractionation of proteins that are encoded by adjacent genes on the chromosome, 106 potential heteromeric PCs containing 243 proteins were identified, only 20 of which were known or expected. In addition to those of unknown function, novel and uncharacterized PCs were identified that are proposed to be involved in the metabolism of amino acids (10), carbohydrates (four), lipids (two), vitamins and metals (three), and DNA and RNA (nine). A further 30 potential PCs were classified as tentative, and the remaining potential PCs (13) were classified as weakly interacting. Some major advantages of native biomass fractionation for PC identification are that it provides a road map for the (partial) purification of native forms of novel and uncharacterized PCs, and the results can be utilized for the recombinant production of low abundance PCs to provide enough material for detailed structural and biochemical analyses.

Virtually all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes, the compositions of which are largely undefined. They cannot be predicted solely from bioinformatics analyses nor are there well defined techniques currently available to unequivocally identify protein complexes (PCs). To address this issue, we attempted to directly determine the identity of PCs from native microbial biomass using Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100 °C, as the model organism. Novel PCs were identified by large scale fractionation of the native proteome using non-denaturing, sequential column chromatography under anaerobic, reducing conditions. A total of 967 distinct P. furiosusproteins were identified by mass spectrometry (nano LC-ESI-MS/MS), representing ∼80% of the cytoplasmic proteins. Based on the co-fractionation of proteins that are encoded by adjacent genes on the chromosome, 106 potential heteromeric PCs containing 243 proteins were identified, only 20 of which were known or expected. In addition to those of unknown function, novel and uncharacterized PCs were identified that are proposed to be involved in the metabolism of amino acids (10), carbohydrates (four), lipids (two), vitamins and metals (three), and DNA and RNA (nine). A further 30 potential PCs were classified as tentative, and the remaining potential PCs (13) were classified as weakly interacting. Some major advantages of native biomass fractionation for PC identification are that it provides a road map for the (partial) purification of native forms of novel and uncharacterized PCs, and the results can be utilized for the recombinant production of low abundance PCs to provide enough material for detailed structural and biochemical analyses.

Rubredoxin from the hyperthermophile Pyrococcus furiosus(Pf Rd) is an extremely thermostable protein, which makes it an attractive subject of protein folding and stability studies. A fundamental question arises as to what the reason for such extreme stability is and how it can be elucidated from a complex set of interatomic interactions. We addressed this issue first theoretically through a computational analysis of the hydrophobic core of the protein and its mutants, including the interactions taking place inside the core. Here we show that a single mutation of one of phenylalanine's residues inside the protein's hydrophobic core results in a dramatic decrease in its thermal stability. The calculated unfolding Gibbs energy as well as the stabilization energy differences between a few core residues follows the same trend as the melting temperature of protein variants determined experimentally by microcalorimetry measurements. NMR spectroscopy experiments have shown that the only part of the protein affected by mutation is the reasonably rearranged hydrophobic core. It is hence concluded that stabilization energies, which are dominated by London dispersion, represent the main source of stability of this protein.