The free-energy relationships found for h-cyt c and y-cyt c folding kinetics imply that the height of the barrier to folding depends upon the relative stabilities of the unfolded and folded states. The striking correspondence in rate/free-energy profiles for h-cyt c and y-cyt c suggests that, despite low sequence homology, they follow similar folding pathways.

The rates of Ru(His33)cytochrome c electron-transfer (ET) reactions have been measured over a driving-force range of 0.59 to 1.89 eV. The driving-force dependence of Fe2+ → Ru3+ ET in RuL2(im)(His33)cyt c [L = 2,2‘-bipyridine (bpy), 4,4‘,5,5‘-tetramethyl-2,2‘-bipyridine (4,4‘,5,5‘-(CH3)4-bpy), 4,4‘-dimethyl-2,2‘-bipyridine (4,4‘-(CH3)2-bpy), 4,4‘-bis(N-ethylcarbamoyl)-2,2‘-bipyridine (4,4‘-(CONH(C2H5))2-bpy), 1,10-phenanthroline (phen); im = imidazole] is well described by semiclassical ET theory with k max = 2.7 × 106 s-1 (HAB = 0.095 cm-1) and λ = 0.74 eV. As predicted by theory, the rate of an exergonic (−ΔG° = 1.3 eV) heme reduction reaction, *Ru2+(bpy)2(im)(His) → Fe3+, falls in the inverted region (k = 2.0 × 105 s-1). In contrast, the rates of three highly exergonic heme reductions, *Ru2+(phen)2(CN)(His) → Fe3+ (2.0 × 105 s-1; 1.40 eV), Ru+(4,4‘-(CONH(C2H5))2-bpy)2(im)(His) → Fe3+ (2.3 × 105 s-1; 1.44 eV), and Ru+(phen)2(CN)(His) → Fe3+ (4.5 × 105 s-1; 1.89 eV), are much higher than expected for reactions directly to ground-state products. Agreement with theory is greatly improved by assuming that an electronically excited ferroheme (Fe2+ → *Fe2+; ∼ 1.05 eV) is the initial product in each of these reactions.

Photochemical techniques have been used to measure the kinetics of intramolecular electron transfer in Ru(bpy)2(im)(His)2(+)-modified (bpy = 2,2'-bipyridine; im = imidazole) cytochrome c and azurin. A driving-force study with the His33 derivatives of cytochrome c indicates that the reorganization energy (lambda) for Fe2+-->Ru3+ ET reactions is 0.8 eV. Reductions of the ferriheme by either an excited complex, *Ru2+, or a reduced complex, Ru+, are anomalously fast and may involve formation of an electronically excited ferroheme. The distance dependence of Fe2+-->Ru3+ and Cu+-->Ru3+ electron transfer in 12 different Ru-modified cytochromes and azurins has been analyzed using a tunneling-pathway model. The ET rates in 10 of the 12 systems exhibit an exponential dependence on metal-metal separation (decay constant of 1.06 A-1) that is consistent with prediction of the pathway model.

The oxidized and reduced forms of a mutant of Pseudomonas aeruginosa azurin, in which the Cys112 has been replaced by an aspartate, have been studied by X-ray absorption spectroscopy. It is well established that the characteristic approximately 600 nm absorption feature of blue copper proteins is due to the S(Cys112) 3ppi --> Cu 3d(x)()()2(-)(y)()()2 charge-transfer transition. While other mutagenesis studies have involved the creation of an artificial blue copper site, the present work involves a mutant in which the native blue copper site has been destroyed, thus serving as a direct probe of the importance of the copper-thiolate bond to the spectroscopy, active site structure, and electron-transfer function of azurin. Of particular interest is the dramatic decrease in electron-transfer rates, both electron self-exchange (k(ese) approximately 10(5) M(-)(1) s(-)(1) wild-type azurin vs k(ese) approximately 20 M(-)(1) s(-)(1) C112D azurin) and intramolecular electron transfer to ruthenium-labeled sites (k(et) approximately 10(6) s(-)(1) wild-type azurin vs k(et)