We have employed the QM(CASPT2//CASSCF)/MM method to explore the excited-state isomerization and decay mechanism of a single-bond-rotation locked photoactive yellow protein (PYP) chromophore in wild-type and mutant proteins. The S1 state is a spectroscopically bright state in the Franck-Condon region. In this state, there exist two excited-state isomerization pathways separately related to the clockwise and anticlockwise rotations of the C=C bond. The clockwise path is favorable because of a small barrier of 2 kcal/mol and uses a novel bicycle-pedal unidirectional photoisomerization mechanism in which the involved two dihedral angles rotate asynchronously because of the reinforced hydrogen-bonding interaction between the chromophore and Cys69. Near the twisted S1 minimum, the chromophore hops to the S0 state via the S1/S0 conical intersection. Finally, the R52A mutation has small effects on the excited-state properties and photoisomerization of the locked PYP chromophore. The present work provides new insights for understanding the photochemistry of PYP chromophores in protein surroundings.