The Mononuclear Metal-Binding Site of Mo-Nitrogenase Is Not Required for Activity.

The biological N2-fixation process is catalyzed exclusively by metallocofactor-containing nitrogenases. Structural and spectroscopic studies highlighted the presence of an additional mononuclear metal-binding (MMB) site, which can coordinate Fe in addition to the two metallocofactors required for the reaction. This MMB site is located 15-Å from the active site, at the interface of two NifK subunits. The enigmatic function of the MMB site and its implications for metallocofactor installation, catalysis, electron transfer, or structural stability are investigated in this work. The axial ligands coordinating the additional Fe are almost universally conserved in Mo-nitrogenases, but a detailed observation of the available structures indicates a variation in occupancy or a metal substitution. A nitrogenase variant in which the MMB is disrupted was generated and characterized by X-ray crystallography, biochemistry, and enzymology. The crystal structure refined to 1.55-Å revealed an unambiguous loss of the metal site, also confirmed by an absence of anomalous signal for Fe. The position of the surrounding side chains and the overall architecture are superposable with the wild-type structure. Accordingly, the biochemical and enzymatic properties of the variant are similar to those of the wild-type nitrogenase, indicating that the MMB does not impact nitrogenase's activity and stability in vitro.

Nitrogen Fixation and Hydrogen Evolution by Sterically Encumbered Mo-Nitrogenase.

The substrate-reducing proteins of all nitrogenases (MoFe, VFe, and FeFe) are organized as α2ß2(γ2) multimers with two functional halves. While their dimeric organization could afford improved structural stability of nitrogenases in vivo, previous research has proposed both negative and positive cooperativity contributions with respect to enzymatic activity. Here, a 1.4 kDa peptide was covalently introduced in the proximity of the P cluster, corresponding to the Fe protein docking position. The Strep-tag carried by the added peptide simultaneously sterically inhibits electron delivery to the MoFe protein and allows the isolation of partially inhibited MoFe proteins (where the half-inhibited MoFe protein was targeted). We confirm that the partially functional MoFe protein retains its ability to reduce N2 to NH3, with no significant difference in selectivity over obligatory/parasitic H2 formation. Our experiment concludes that wild-type nitrogenase exhibits negative cooperativity during the steady state regarding H2 and NH3 formation (under Ar or N2), with one-half of the MoFe protein inhibiting turnover in the second half. This emphasizes the presence and importance of long-range (>95 Å) protein-protein communication in biological N2 fixation in Azotobacter vinelandii.

Evaluation of pediatric lateral oropharyngeal trauma.

OBJECTIVE: We reviewed the mechanism of injury, presentation, and evaluation of children with trauma to the lateral oropharynx. Study design and setting We conducted a retrospective review of patients in an urban pediatric emergency department with trauma to the lateral oropharynx over a 5-year period. RESULTS: Forty-eight patients were identified with documented injuries of the lateral oropharynx placing the internal carotid artery at risk of injury. The average age was 42 months, with a male-to-female ratio of 1.5:1. Seventy-seven percent of patients had a documented neurologic examination. Examinations were normal in all cases. Computed tomography scans were obtained in 14 patients, identifying 3 patients with carotid abnormalities. Angiography subsequently diagnosed intimal injuries in 2 of the 3 patients. There were no known cases of neurovascular compromise. CONCLUSION: Contrast-enhanced computed tomography may be an effective screening examination in this patient population, helping to determine which children should be admitted for angiography and observation.