Fungal cellobiose dehydrogenases (CDHs) are divided on the basis of amino acid sequence into class I (from basidiomycetes) and class II (from ascomycetes), which show quite different pH-dependence of the activity. Here, we mutated glutamine 734 (Q734) in the flavin domain of class I CDH from the basidiomycete Phanerochaete chrysosporium to the corresponding amino acid in class II CDHs (serine or threonine), and compared the kinetics of the mutant enzymes (Q734S and Q734T) and wild-type enzyme (WT). The two mutant enzymes showed almost identical absorption spectra, although that of WT was slightly different. When the steady-state kinetic parameters were compared at pH 4.0 and 7.0, WT showed the highest activity at both pH values. However, k _cat/ K _m for Q734S was similar at both pHs, whereas k _cat/ K _m for Q734T was 3.5 times higher at pH 4.0 than that at pH 7.0. As for the pH-dependence of the specific activity, Q734S did not have an apparent optimum pH in the pH range tested, whereas Q734T showed an acidic optimum pH profile compared with WT. These differences can be explained in terms of the effect of the side chain of the amino acid residue at position 734 on the reactivity of the flavin cofactor.