Stopped-Flow Fourier Transform Infrared Spectroscopy Allows Continuous Monitoring of Azide Reduction, Carbon Monoxide Inhibition, and ATP Hydrolysis by Nitrogenase

• Published in: Biochemistry (Easton) Vol. 44; no. 27; pp. 9520 - 9527
• Main Authors: Tolland, John D, Thorneley, Roger N. F
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 12.07.2005
• Subjects: Adenosine Diphosphate - chemistry; Adenosine Monophosphate - chemistry; Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; Azides - antagonists & inhibitors; Azides - chemistry; Binding Sites; Carbon Monoxide - chemistry; Carbon Monoxide - metabolism; Hydrolysis; Kinetics; Klebsiella pneumoniae - enzymology; Magnesium - chemistry; Models, Chemical; Molybdoferredoxin - chemistry; Molybdoferredoxin - metabolism; Nitrogenase - chemistry; Nitrogenase - metabolism; Oxidation-Reduction; Phosphates - chemistry; Protein Binding; Spectroscopy, Fourier Transform Infrared - methods; Substrate Specificity; Time Factors
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi050453m

Stopped-flow FTIR spectroscopy was used to monitor continuously the pre-steady- and steady-state phases of azide reduction by nitrogenase and the accompanying hydrolysis of ATP. This was characterized by a ca. 1.3 s lag phase that is explained by the number of Fe protein cycles required to effect the reductions of azide to N2 + NH3, N2H4 + NH3, or 3NH3. Extrapolation of the steady-state time course for azide reduction to zero time showed that one azide binds within 200 ms to each FeMo cofactor. Inhibition of azide reduction by CO was established at times <400 ms, which was faster than the appearance of the first observable IR band assigned to CO (1904 cm-1 detectable at ca. 1 s with maximum amplitude at ca. 7 s). IR bands associated with the rapidly formed (<400 ms) CO species that inhibits azide reduction were not observed over the range 1700−2100 cm-1. This suggests either that the CO is initially bridging two or more Fe atoms or that a rapid reduction of CO to a formyl state occurs by insertion into a metal−hydride bond. The frequencies and time courses for the appearance and loss of the CO bands under hi- and lo-CO conditions were essentially unaffected by the presence of 20 mM azide, consistent with CO being a noncompetitive inhibitor of azide reduction and with azide and CO binding to different sites on the FeMo cofactor.