Atomic resolution modeling of the ferredoxin:[FeFe] hydrogenase complex from Chlamydomonas reinhardtii

Abstract

The [FeFe] hydrogenases HydA1 and HydA2 in the green alga Chlamydomonas reinhardtii catalyze the final reaction in a remarkable metabolic pathway allowing this photosynthetic organism to produce H(2) from water in the chloroplast. A [2Fe-2S] ferredoxin is a critical branch point in electron flow from Photosystem I toward a variety of metabolic fates, including proton reduction by hydrogenases. To better understand the binding determinants involved in ferredoxin:hydrogenase interactions, we have modeled Chlamydomonas PetF1 and HydA2 based on amino-acid sequence homology, and produced two promising electron-transfer model complexes by computational docking. To characterize these models, quantitative free energy calculations at atomic resolution were carried out, and detailed analysis of the interprotein interactions undertaken. The protein complex model we propose for ferredoxin:HydA2 interaction is energetically favored over the alternative candidate by 20 kcal/mol. This proposed model of the electron-transfer complex between PetF1 and HydA2 permits a more detailed view of the molecular events leading up to H(2) evolution, and suggests potential mutagenic strategies to modulate electron flow to HydA2.

FIGURE 1

(A) The thermodynamic cycle considered, with the calculation methods for each step shown. Associated shaded text implies calculation of energy for the individual protein component (solid text) from the dynamics trajectory of the docked complex. Vibrational free energy changes were calculated from molecular dynamics runs on the free proteins. (B) Model complex 16. (C) Model complex 42.

FIGURE 2

Sequence alignment of selected [2Fe-2S] ferredoxins. Numbering of residues identified in the literature has been adjusted to match the sequences shown. Residues are highlighted to denote those experimentally identified as located at active electron transfer interfaces (yellow), and at the complex 16 (red), 42 (light blue), or 16 and 42 (light purple) interfaces in this study. C. reinhardtii residues in bold denote those interacting with nitrite reductase and glutamate synthase (90). Absolute and partial identities and functional conservation are denoted underneath the sequence blocks with symbols {*, .,:}, respectively.

FIGURE 3

Interaction surfaces of complexes 16 (A) and 42 (B), color-coded by residue interactions. Hydrogenase HydA2 is oriented similarly in the two images for comparison, and the ferredoxin has been omitted for clarity. (Colors are: red, like charges; orange, charge-polar; yellow, aromatic-aromatic; green, charge-aliphatic; blue, aliphatic-aliphatic; gray, opposite charges.)

FIGURE 4

Timecourse of closest S–S approach distance between electron transfer cofactors on ferredoxin and hydrogenase HydA2 during molecular dynamics simulations for (A) complex 16, and (B) complex 42.

FIGURE 5

Plot of representative reduced-basis quasiharmonic analysis (top), and residuals (solid circles, bottom). Data (solid squares) were fit to a biexponential equation (line), and configurational entropy calculated by extrapolation of this equation to infinite time.

FIGURE 6

Electrostatic isopotential surfaces for ferredoxin (top) and HydA2 (bottom), contoured at +1.0 k_BT/q_e (blue) and −1.0 k_BT/q_e (red). Pictures on the left represent direct views of the protein-protein interface; those on the right are rotated by 90° clockwise about the vertical axis running through the protein and in the plane of the figure.