This paper describes methods of constrained and restrained refinement of EXAFS data which provide a means of substantially reducing the number of independent parameters compared to conventional least-squares methods commonly used. Constrained refinement allows a major reduction in the number of free parameters for a refinement of a structural model. In restrained refinement, additional structural information from well-characterized small molecules is used to provide additional observations in the data analysis. Even though these methods are of general application to the majority of complex systems, they are particularly valuable for biological molecules. The methods are of major advantage for ligands where significant multiple scattering is present, e.g., histidine, tyrosine, CO, CN, etc. The bases of these methods are described, and applications to some complex chemical and biological systems are given.

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The reduction of nitrite (NO 2 ؊ ) into nitric oxide (NO), catalyzed by nitrite reductase, is an important reaction in the denitrification pathway. In this study, the catalytic mechanism of the copper-containing nitrite reductase from Alcaligenes xylosoxidans (AxNiR) has been studied using single and multiple turnover experiments at pH 7.0 and is shown to involve two protons. A novel steady-state assay was developed, in which deoxyhemoglobin was employed as an NO scavenger. A moderate solvent kinetic isotope effect (SKIE) of 1.3 ؎ 0.1 indicated the involvement of one protonation to the rate-limiting catalytic step. Laser photoexcitation experiments have been used to obtain single turnover data in H 2 O and D 2 O, which report on steps kinetically linked to inter-copper electron transfer (ET). In the absence of nitrite, a normal SKIE of ϳ1.33 ؎ 0.05 was obtained, suggesting a protonation event that is kinetically linked to ET in substratefree AxNiR. A nitrite titration gave a normal hyperbolic behavior for the deuterated sample. However, in H 2 O an unusual decrease in rate was observed at low nitrite concentrations followed by a subsequent acceleration in rate at nitrite concentrations of >10 mM. As a consequence, the observed ET process was faster in D 2 O than in H 2 O above 0.1 mM nitrite, resulting in an inverted SKIE, which featured a significant dependence on the substrate concentration with a minimum value of ϳ0.61 ؎ 0.02 between 3 and 10 mM. Our work provides the first experimental demonstration of proton-coupled electron transfer in both the resting and substrate-bound AxNiR, and two protons were found to be involved in turnover.