Structure of C73G putidaredoxin fromPseudomonas putida

Abstract

The structure of the C73G mutant of putidaredoxin (Pdx), the Fe(2)S(2) ferredoxin that supplies electrons to cytochrome CYP101 (p450cam) for camphor oxidation, is reported at 1.9 A resolution in a C2 crystal form. The structure was solved by single-wavelength iron anomalous diffraction, which yielded electron density above the 2sigma level for over 97% of the non-H atoms in the protein. The final structure with R = 0.19 and R(free) = 0.21 has been deposited in the Protein Data Bank with accession code 1r7s. The C2 crystal contains three Pdx molecules in the asymmetric unit, giving three independent models of the protein that are very similar (r.m.s.d. < 0.3 A for the 106 C(alpha) atoms). The unusually high solvent fraction of 80% results in comparatively few crystal-packing artifacts. The structure is briefly compared with the recently reported crystal structures of the C73S and C73S/C85S mutants. In general, the eight independent molecules in the three crystal structures (three in C73G, three in C73S and two in C73S/C85S) are much more similar to each other than to the previously reported NMR structure of wild-type Pdx in solution. The present findings show a unanimous structure in some regions crucial for electron-transfer interactions, including the cluster-binding loop 39-48 and the cytochrome-interaction region of Asp38 and Trp106. In addition, the Cys45 amide group donates a hydrogen bond to cluster sulfur S1, with Ala46 adopting an Lalpha conformation, in all three molecules in the crystal.

Redox-dependent Structural Reorganization in Putidaredoxin, a Vertebrate-type [2Fe-2S] Ferredoxin from Pseudomonas putida

“…This proposal contradicts the fact that there are a few crystal structures of oxidized [2Fe-2S] ferredoxins or ferredoxin-like domains where the carbonyl oxygen atom of the corresponding cysteinyl ligand is in the CO out conformation. 15,[27][28][29][30] Thus, more structural data are needed to understand how the iron-sulfur proteins stabilize the reduced form and how redox conformational changes affect redox partner recognition.…”

“…31 The previously reported crystal structures of the oxidized C73S, C73S/C85S, and C73G Pdx mutants were solved using synchrotron radiation as an X-ray source. 16,30 As singlecrystal X-ray absorption spectroscopy experiments indicate, crystalline Pdx can be photoreduced to some extent by synchrotron radiation. 16 The reported crystallographic models, therefore, may not be representative of the fully oxidized ironsulfur proteins.…”

Crystal Structure of the Putidaredoxin Reductase·Putidaredoxin Electron Transfer Complex

“…Acting as a shuttle, Pdx forms transient electron transfer (ET) complexes with its redox partners during turnover (2)(3)(4)(5)(6). The x-ray structures of all components of the camphor monooxygenase have been determined (7)(8)(9)(10), but neither Pdr⅐Pdx nor Pdx⅐P450 cam native complexes have been crystallized thus far.…”

Putidaredoxin-to-Cytochrome P450cam Electron Transfer: Differences between the Two Reductive Steps Required for Catalysis

“…However, the 1PUT Pdx structure (25) used by Pochapsky' group for model building differs significantly from the high-resolution x-ray structures (26,27) and a refined solution model of the iron-sulfur protein (28). Another limitation on the P450cam-Pdx model is that both protein structures are in the oxidized state.…”