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Reorganization and conformational changes in the reduction of tetraheme cytochromes.

ABSTRACT: Molecular dynamics simulation (MD) constitutes an alternative to time-consuming experiments for studying conformational changes. We apply MD on a redox system where experimental information exists for the fully oxidized and fully reduced states: tetraheme cytochrome c3. Instead of doing one simulation for each state, we apply 10 4-ns replicas for both states, which provides robust statistics to characterize the redox changes. Besides these long simulations, we perform 120 short ones (50 ps), where an equilibrated oxidized state is perturbed to a reduced state. This allows the application of a nonequilibrium method, the subtraction technique, which makes it possible to characterize the different timescales of conformational changes. Reduction induces conformational changes in the N-terminus and on the loops spanning residues 36-42 and 88-93, which correlate very well with experiments, demonstrating the applicability of this methodology. We also analyze the effect of reduction on hydrogen bonds, solvent accessible surface and bound water, the changes being found to involve the hemes and propionate groups. Redox-induced protonation is also investigated, by protonating the propionates D from hemes I and IV. Although this change in the former does not have major conformational consequences, it induces in the latter conformational changes beyond the ones obtained with reduction.

SUBMITTER: Oliveira AS

PROVIDER: S-EPMC1366959 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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Reorganization and conformational changes in the reduction of tetraheme cytochromes.

Oliveira A Sofia F AS Teixeira Vitor H VH Baptista António M AM Soares Cláudio M CM

Biophysical journal 20050916 6

Molecular dynamics simulation (MD) constitutes an alternative to time-consuming experiments for studying conformational changes. We apply MD on a redox system where experimental information exists for the fully oxidized and fully reduced states: tetraheme cytochrome c3. Instead of doing one simulation for each state, we apply 10 4-ns replicas for both states, which provides robust statistics to characterize the redox changes. Besides these long simulations, we perform 120 short ones (50 ps), whe ...[more]

PMID: 16169983

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Project description:This Account summarizes recent findings centered on the role that redox partner binding, allostery, and conformational dynamics plays in cytochrome P450 proton coupled electron transfer. P450s are one of Nature's largest enzyme families and it is not uncommon to find a P450 wherever substrate oxidation is required in the formation of essential molecules critical to the life of the organism or in xenobiotic detoxification. P450s can operate on a remarkably large range of substrates from the very small to the very large, yet the overall P450 three-dimensional structure is conserved. Given this conservation of structure, it is generally assumed that the basic catalytic mechanism is conserved. In nearly all P450s, the O2 O-O bond must be cleaved heterolytically enabling one oxygen atom, the distal oxygen, to depart as water and leave behind a heme iron-linked O atom as the powerful oxidant that is used to activate the nearby substrate. For this process to proceed efficiently, externally supplied electrons and protons are required. Two protons must be added to the departing O atom while an electron is transferred from a redox partner that typically contains either a Fe2S2 or FMN redox center. The paradigm P450 used to unravel the details of these mechanisms has been the bacterial CYP101A1 or P450cam. P450cam is specific for its own Fe2S2 redox partner, putidaredoxin or Pdx, and it has long been postulated that Pdx plays an effector/allosteric role by possibly switching P450cam to an active conformation. Crystal structures, spectroscopic data, and direct binding experiments of the P450cam-Pdx complex provide some answers. Pdx shifts the conformation of P450cam to a more open state, a transition that is postulated to trigger the proton relay network required for O2 activation. An essential part of this proton relay network is a highly conserved Asp (sometimes Glu) that is known to be critical for activity in a number of P450s. How this Asp and proton delivery networks are connected to redox partner binding is quite simple. In the closed state, this Asp is tied down by salt bridges, but these salt bridges are ruptured when Pdx binds, leaving the Asp free to serve its role in proton transfer. An alternative hypothesis suggests that a specific proton relay network is not really necessary. In this scenario, the Asp plays a structural role in the open/close transition and merely opening the active site access channel is sufficient to enable solvent protons in for O2 protonation. Experiments designed to test these various hypotheses have revealed some surprises in both P450cam and other bacterial P450s. Molecular dynamics and crystallography show that P450cam can undergo rather significant conformational gymnastics that result in a large restructuring of the active site requiring multiple cis/trans proline isomerizations. It also has been found that X-ray driven substrate hydroxylation is a useful tool for better understanding the role that the essential Asp and surrounding residues play in catalysis. Here we summarize these recent results which provide a much more dynamic picture of P450 catalysis.

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Reorganization and conformational changes in the reduction of tetraheme cytochromes.

Publications

Reorganization and conformational changes in the reduction of tetraheme cytochromes.

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