Characterization of Conformational Changes Coupled to Ligand Photodissociation from the Heme Binding Domain of FixL

Abstract

Using transient absorption spectroscopy and photoacoustic calorimetry (PAC), we have characterized carbon monoxide photodissociation and rebinding to two forms of the heme domain of Bradyrhizobium japonicum FixL. Transient absorption results for the complete heme domain (FixL residues 140-270) and a truncated heme domain (missing 11 residues on the N-teminal end and 14 amino acid residues on the C-terminal end of the full length heme domain) show similar rates for ligand rebinding to the five-coordinate heme domain and the absence of any transient intermediate on a microsecond time scale. Results from PAC studies show that both the truncated and complete heme domains undergo a contraction upon ligand photolysis. In addition, CO photolysis from the complete heme domain gives rise to an intermediate with a lifetime of approximately 150 ns which is absent in the truncated heme domain. We attribute the 150 ns phase to ligand release to the solvent which may be accelerated in the case of the truncated domain. The initial contraction is attributed to changes in the charge distribution due to reorganization of the surface salt bridge formed between Glu182 and Arg227 or possibly to reorientation of Arg206. Changes in the charge distribution may play an important role in communication between the sensor domain and the regulatory domain and thus may be part of the signal transduction pathway.

Photoacoustic characterization of protein dynamics following CO photodetachment from fully reduced bovine cytochrome c oxidase

“…In this case, the enthalpic changes for resolvable reactions steps, which occur subsequent to prompt heat release are described as DH i = Àq i . This is how Di Primo et al [40][41][42] reported DH values for CO photodetachment from cytochrome P450 cam and how Larsen and coworkers report their results [44][45][46][47][48][49][50][51][52]. Because we will compare our results to theirs, we follow this precedent in Section 4.…”

“…We speculate that this conformational change involves the displacement of one or more relatively exposed charges inward, out of contact with solvent, as would occur in the closing of a salt bridge, and that this motion is coupled to changes in ligand mobility with the heme module of the protein. In view of other studies [38,41,52,65], similar conformational changes may be common to heme proteins, and photoacoustic approaches will be useful in their characterization. The photoacoustic signals from cytochrome oxidase systems are particularly complex as they arise from a variety of events following CO photolysis, they seem to be dependent on buffer composition, and protocols to facilitate their resolution and deconvolution will be necessary.…”

“…We will use this terminology in our initial analyses (see Tables 1 and 2, below) but will discontinue it when exploring effects of ionic strength on the thermodyamic parameters (Table 3, below), to facilitate comparison to results reported for the other heme proteins [40][41][42][44][45][46][47][48][49][50][51][52]. Thus to avoid confusion, we have explicitly defined our terminology for the enthalpic parameters here.…”

“…Larsen and coworkers have applied photothermal techniques to study CO photodetachment from heme-copper oxidases [44][45][46], model compounds [47][48][49], and smaller heme proteins [50][51][52]. They used photoacoustic measurements on both cytochrome bo 3 from E. coli [44,46] and bovine cytochrome c oxidase (cytochrome aa 3 ) [45].…”

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

“…In contrast to optical techniques, such as transient absorption and IR spectroscopy, that monitor the time profile of absorption changes in the chromophore surrounding; photothermal techniques detect the time-dependence of heat/volume changes and therefore are valuable tools for investigating time profiles of optically "silent" processes. So far, photoacoustic calorimetry and transient grating has been successfully applied to study conformational dynamics of photo-induced processes including diatomic ligand migration in globins 3,4 , ligand interactions with oxygen sensor protein FixL 5 , electron and proton transport in heme-copper oxidases 6 and photosystem II as well as photo-isomerization in rhodopsin 7 and conformational dynamics in cryptochrome 8 .…”