The bacterial flagellar filament is a helical propeller constructed from 11 protofilaments of a single protein, flagellin. The filament switches between left- and right-handed supercoiled forms when bacteria switch their swimming mode between running and tumbling. Supercoiling is produced by two different packing interactions of flagellin called L and R. In switching from L to R, the intersubunit distance ( approximately 52 A) along the protofilament decreases by 0.8 A. Changes in the number of L and R protofilaments govern supercoiling of the filament. Here we report the 2.0 A resolution crystal structure of a Salmonella flagellin fragment of relative molecular mass 41,300. The crystal contains pairs of antiparallel straight protofilaments with the R-type repeat. By simulated extension of the protofilament model, we have identified possible switch regions responsible for the bi-stable mechanical switch that generates the 0.8 A difference in repeat distance.

The iron-containing nitrile hydratase (NHase) is a photoreactive enzyme that is inactivated in the dark because of persistent association with NO and activated by photo-dissociation of NO. The crystal structure at 1.7 A resolution and mass spectrometry revealed the structure of the non-heme iron catalytic center in the nitrosylated state. Two Cys residues coordinated to the iron were post-translationally modified to Cys-sulfenic and -sulfinic acids. Together with another oxygen atom of the Ser ligand, these modifications induced a claw setting of oxygen atoms capturing an NO molecule. This unprecedented structure is likely to enable the photo-regulation of NHase and will provide an excellent model for designing photo-controllable chelate complexes and, ultimately, proteins.

The bacterial flagellum is a motile organelle, and the flagellar hook is a short, highly curved tubular structure that connects the flagellar motor to the long filament acting as a helical propeller. The hook is made of about 120 copies of a single protein, FlgE, and its function as a nano-sized universal joint is essential for dynamic and efficient bacterial motility and taxis. It transmits the motor torque to the helical propeller over a wide range of its orientation for swimming and tumbling. Here we report a partial atomic model of the hook obtained by X-ray crystallography of FlgE31, a major proteolytic fragment of FlgE lacking unfolded terminal regions, and by electron cryomicroscopy and three-dimensional helical image reconstruction of the hook. The model reveals the intricate molecular interactions and a plausible switching mechanism for the hook to be flexible in bending but rigid against twisting for its universal joint function.

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a novel enzyme that possesses a non-heme iron(III) center binding endogenous nitric oxide (NO). It is inactivated by aerobic incubation of cells in the dark, whereas the inactive form is converted to the active one by light irradiation. To clarify the mechanism of activity regulation in the NHase, we investigated the role of NO. When the inactive NHase was irradiated in the presence of Fe(II) ions and a spin trap, N-methyl-D-glucamine dithiocarbamate (MGD), three hyperfine lines from the nitrogen atom of the [(MGD) 2 -Fe II -NO] complex were resolved, indicating that NO was released from the enzyme upon photoactivation. The amount of NO release was obtained as 0.99 ( 0.12 per enzyme (n ) 4) by using an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide. The active NHase was completely inactivated by exogenous NO, and upon photoirradiation 86% of the original activity was restored. FTIR, ESR, and UV-VIS absorption measurements confirmed the view that association of NO restores the original inactive form of the enzyme. The rate constant for nitrosylation of the enzyme activated by a laser pulse asymptotically increased with an increase in NO concentration. A kinetic analysis of the NHase nitrosylation demonstrated that inactivation of the enzyme by NO binding proceeded via an intermediate. The rate constant for inactivation and the quantum yield of photoactivation were determined as 14 s -1 and 0.48, respectively. It is thus concluded that the activity of the NHase is regulated by nitrosylation and photoinduced denitrosylation of the non-heme iron center. This finding provides a new aspect regarding biological function of NO, i.e., regulation of the enzymatic activity with the aid of light.

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Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated by nitrosylation of the non-heme iron center and activated by photodissociation of nitric oxide (NO). To obtain structural information on the iron center, we isolated peptide complexes containing the iron center by proteolysis. When the tryptic digest of the ␣ subunit isolated from the inactive form was analyzed by reversed-phase high performance liquid chromatography, the absorbance characteristic of the nitrosylated iron center was observed in the peptide fragment, Asn 105 -Val-Ile-Val-CysSer-Leu-Cys-Ser-Cys-Thr-Ala-Trp-Pro-Ile-Leu-Gly-LeuPro-Pro-Thr-Trp-Tyr-Lys 128 . The peptide contained 0.79 mol of iron/mol of molecule as well as endogenous NO. Subsequently, by digesting the peptide with thermolysin, carboxypeptidase Y, and leucine aminopeptidase M, we found that the minimum peptide segment required for the nitrosylated iron center is the 11 amino acid residues from ␣Ile 107 to ␣Trp 117 . Furthermore, by using mass spectrometry, protein sequence, and amino acid composition analyses, we have shown that the 112th Cys residue of the ␣ subunit is post-translationally oxidized to a cysteine-sulfinic acid (Cys-SO 2 H) in the NHase. These results indicate that the NHase from Rhodococcus sp. N-771 has a novel non-heme iron enzyme containing a cysteine-sulfinic acid in the iron center. Possible ligand residues of the iron center are discussed.Nitrile hydratase (NHase; EC 4.2.1.84) 1 is a bacterial metalloenzyme catalyzing the hydration of nitriles to corresponding amides (1, 2). NHase consists of two kinds of subunits (␣ and ␤ with the molecular mass values of 23 kDa) and contains non-heme iron (3) or non-corrinoid cobalt (4) atoms. The NHase from Rhodococcus sp. N-771, which is a ␣␤ heterodimer containing a non-heme iron, is inactivated by aerobic incubation in the dark for a half-day (dark-inactivation), whereas the enzyme purified from the dark-inactivated cells is immediately converted to the active form by light irradiation (photoactivation) (5, 6). Recently, it has been revealed that dark-inactivation and photoactivation are controlled by association and photodissociation of nitric oxide (NO) with the non-heme iron center (7-9). Similar photosensitivity is observed in the NHases from Rhodococcus sp. N-774 (10) and R312.2 These NHases seem to be identical to the one from Rhodococcus sp. N-771 because of the same nucleotide sequences (11, 12). 3The iron-containing NHase is the first enzyme with a mononuclear low-spin non-heme iron(III) which is thought to be involved in the catalysis (3). The structure of the iron center in the active form has been studied by various spectroscopies including ESR (3), resonance Raman (13), extended x-ray absorption fine structure (13) and electron nuclear double resonance (14), and the ligand-donor set of N 3 OS 2 has been proposed (14), which is supported by model complexes of the iron center (15-17). Recently, the metal site structure has been studied in detail by means of electro...

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Nitrile hydratase (NHase) from Rhodococcus sp. N-771 exists in active and inactive forms. The inactive NHase is immediately activated by light irradiation and changes to the active form. To characterize the photoreactive center, the inactive NHase was denatured by 6 M urea, and two kinds of subunits (alpha and beta) were separated and purified by anion-exchange chromatography. In a manner similar to the native NHase, the isolated alpha subunit showed two absorption peaks at 280 and 370 nm, which were diminished by light irradiation. However, irradiation failed to elicit the appearance of absorption peaks at around 400 nm and at 710 nm, which were characteristic of the activated enzyme. The beta subunit seemed not to possess any photoreactive chromophore because its absorption spectrum was not altered by light irradiation. Neither of the subunits showed NHase activity before and after light irradiation, but the inactive NHase was reconstituted by incubating the two subunits together in the dark at 4 degrees C for 1 h. Light irradiation of the beta subunit did not affect subsequent complex formation or NHase activity. However, the irradiated alpha subunit could not assemble with the beta subunit, and no activity was recovered. These results demonstrate that the chromophore(s) responsible for the photoactivation of NHase are entirely located on the alpha subunit, and imply that light irradiation induces conformational change of the alpha subunit.