* To whom correspondence should be addressed.
Received December 22, 1997; Revision received April 28, 1998
The ability of xanthene dyes to mediate photoinduced reduction of nitrogenase was tested. In addition to eosin, which was studied in the preceding work (Biochemistry (Moscow), 1996, 61, 2165-2172), 4´,5´-dibromofluorescein (DBF), cyanosine, and erythrosin are effective photodonors of an electron in the presence of NADH. Fluorescein, rhodamine B, rhodamine 6G, and porphyrins are unable to mediate photoinduced reduction of nitrogenase. The mechanism underlying different efficiency of xanthene dyes in this reaction was studied. At high concentrations, all xanthene dyes tested were shown to inhibit the intramolecular electron transfer in nitrogenase. The inhibiting concentration of DBF is 1.5·10-4 M, whereas for other dyes, the inhibiting concentrations are less than 1.5·10-4 M. Under otherwise identical conditions, the ATPase activity was inhibited by xanthene dyes to a lesser extent than the nitrogenase activity. DBF, the most effective photodonor, was also studied by differential kinetic pulse laser spectroscopy. Photoinduced reduction of nitrogenase, (Fe-proteinox·Mo-Fe-protein)·MgATP or (Av2ox·Av1)·MgATP, was studied within the time range from 0 to 100 msec. Two initial stages of the nitrogenase turnover were detected: photoinduced reduction of Av2 and electron transfer from Av2red to Av1. The kinetics of the photoinduced reduction of Av2·MgADP was studied in the presence of DBF (up to 1.3·10-4 M) both in solution and the complex with Av1. The apparent second-order rate constants of the photoinduced reduction of Av2·MgADP in solution and the complex with Av1 were determined as 9.7·107 ± 106 and 1.2·108 ± 1.2·107 M-1·sec-1, respectively. The rate constant of the second reaction in the presence of another donor (dithionite) is 2500 times less. In complexes with Av1, the photochemical donor system DBF--NADH reduces Av2 more effectively than in free state in solution. In the presence of the photochemical donor system, neither photoreduction of Av2 in complexes with Av1 nor electron transfer from Av2red to Av1 are the rate-limiting stages of nitrogenase turnover.
KEY WORDS: nitrogenase, Fe-protein, Mo-Fe-protein, kinetic laser spectroscopy, differential pulse spectroscopy